Polynucleotide encoding a novel human serpin secreted from lymphoid cells, LSI-01

ABSTRACT

The present invention provides novel polynucleotides encoding LSI-01 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel LSI-01 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

This application claims benefit to provisional application U.S. Ser. No. 60/248,434, filed Nov. 14, 2000; to provisional application U.S. Ser. No. 60/257,610, filed Dec. 21, 2000; and to provisional application U.S. Ser. No. 60/282,745, filed Apr. 10, 2001.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides encoding LSI-01 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel LSI-01 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

BACKGROUND OF THE INVENTION

Limited-proteolysis by serine and cysteine proteases plays a central regulatory role in many physiological and pathophysiological processes including coagulation, fibrinolysis, complement fixation, apoptosis, angiogenesis, tumor cell attachment and growth. Serpins (Serine Protease Inhibitors) are the principle protease inhibitors in human plasma (serpins make up 10% of plasma proteins on a molar basis), and have been shown to regulate these physiological processes. Well known examples include antithrombin which regulates the blood coagulation cascade; C₁-inhibitor which controls complement activation; plasminogen activator inhibitors (PAI-1 and α₂-antiplasmin) which regulate fibrinolysis; and alpha₁-antitrypsin, also called alpha₁ proteinase inhibitor, which modulates connective tissue remodeling (see Gils & Declerck, 1998 and Whisstock et al. 1998 for reviews on these and other serpins).

Serpins regulate proteases through a suicide-substrate inhibition mechanism forming a covalent complex between the protease active site serine and the bait P1 amino acid (Huntington et al., 2000). This results in a 1:1, SDS-resistant complex. In addition to the inhibitory serpins, there are also serpin family members that function as hormone transport or growth factor proteins (examples include the thyroxin and corticosteroid binding globulins and the vasopressor peptide source angiotensinogen). In addition, serpins have demonstrated involvement in angiogenesis and tumor growth (see for example O'Reilly et al., 1999).

As a class, serpins contain conserved residues located internal to the protein or on surface niches, therefore the serpin family has remarkably-conserved 3-dimentional structure despite less dramatic overall homology elsewhere (see Huber & Carrell,1989 and Whisstock et al., 1998 for reviews). Protease inhibitory serpins (of which the present invention relates) contain a mobile reactive site loop (P16-P10′) that is subject to proteolytic cleavage. This loop cleavage site (sometimes referred to as the “bait region”) is cleaved by a cognate protease. The loop region is flexible and has the ability to profoundly change its conformation (alanine-rich regions in the loop P13-P9 allow increased flexibility (Gils & Declerck, 1998)). Once cleaved, the P14-P1 portion of the loop has high affinity for insertion into a β-sheet domain (Whisstock et al., 1998; Huntington et al., 2000). In general, peptides corresponding to the P14-P1 or P14-P7 or P14-P10 loop regions and small molecule mimics of the loop region prevent protease inhibitory activity.

The serpin proteolytic cleavage site is designated P1P1′. It is generally accepted that the P1 residue is the major determinant of the protease specificity of the serpin (Gills & Declerck, 1998). Typically the bait region (especially the P1 site) mimics the natural substrate of the cognate protease. While each inhibitory serpin shows some preference for selected proteases, most serpins recognize several proteases with varying degrees of specificity (for example antithrombin will inhibit Factors IXa, Xa, XIa, as well as thrombin; α1-antitrypsin will inhibit trypsin and neutrophil elastase; PAI-1 inhibits tPA, uPA and trypsin etc.). Furthermore, relatively minor amino acid changes in the bait region, especially at the P1 position, can dramatically change the specificity of the serpin (Gils and Declerck, 1998).

In addition to the bait region, domains away from the cleavage site also confer selectivity. For some serpins, non-enzymatic cofactors activate the serpin and modulate the rate of protease inactivation. For example antithrombin, PAI-1, protease nexin 1, protein C inhibitor and heparin cofactor II are stimulated by heparin. Furthermore, the specificity of a serpin may depend on the cofactor composition and concentration. For example the specificity of antithrombin depends on the size of the heparin molecule. Large molecular weight heparins stimulate inhibition of thrombin where as low molecular heparin has more FXa selectivity. Another example is vitronectin, which binds to PAI-1 and increases its affinity for thrombin. Studies (Shirk, et al, 1994, Lane et al, 1994, Pratt et al 1991, Blinder, et al, 1989, Blinder et al 1991, Whinna et al, 1991, Ragg et al, 1991) have implicated one or more of the A, D and H helices of serpins (for example, the helices in antithrombin, protein C inhibitor and heparin cofactor II) which are rich in basic residues and bind heparin, and, 3-dimensional structures and models have been useful in the design of low molecular weight molecules which modulate serpin reactivity.

Recently, the crystal structure of a serpin-protease complex was reported (Huntington, et al, 2000) in which the reactive loop has been cleaved at the bait position. The tethered protease moves 70 angstroms, with the tether sequence (P10-P1) inserted in the “A” beta sheet of alpha anti-trypsin. This shallow groove, which we call the cleaved reactive loop-binding region, represents a potential target for small molecule inhibition.

Serpins in Disease and Therapy

Members of the serpin family play a variety of roles in physiology, disease and in therapy. Deficiencies in some serpins can cause well-characterized diseases. Alpha-1-antitrypsin deficiency, which is a common autosomal recessive disorder, is associated with development of emphysema, liver cirrhosis and hepatocellular carcinoma. Patients deficient in antithrombin are prone to sever thrombotic consequences.

Serpins are useful as therapeutics. Antithrombin is a potent inhibitor of thrombin-mediated vascular injury in the microcirculation in severe sepsis. This endogenous anticoagulant is rapidly depleted in the early phases of sepsis as a result of decreased synthesis, increased destruction, and enhanced clearance by thrombin-antithrombin complex formation. The therapeutic efficacy of antithrombin in experimental sepsis is readily demonstrable in numerous animal systems (Opal 2000).

In addition to the therapeutic activity of the serpins, modulators of serpin activity have well documented utility in antithrombotic therapy. The anticoagulant efficacy of heparin and low molecular weight heparin is mediated by antithrombin inhibition of proteases (i.e. thrombin and FXa). Indeed antithrombin is required for the anti-coagulant efficacy of heparin and low molecular weight heparins which are useful in the treatment of arterial thrombosis (for example myocardial infarction, unstable angina & stroke) and venous thrombosis (for example deep vein thrombosis and pulmonary embolism).

Inhibitors of serpins have also shown utility. Antibodies or small molecule inhibitors can inhibit serpin activity and prolong cognate protease function. Examples of serpin inhibition with therapeutic value that preserve fibrinolytic function include antibodies to alpha 2-antiplasmin (Reed et al., 1990) and plasminogen activator inhibitor-1 (Biemond et al., 1995). Both have been shown to improve thrombolysis under thrombotic conditions in animal models of disease.

Serpins are also useful in measurement of cognate protease activation and are useful in diagnosis of disease states. In general, proteolytic enzymes form stable complexes with serpins that can be followed by measuring the cognate protease-serpin complex using ELISA or other techniques. As such, measurement of the serpin-protease complex is useful as a reporter of cognate protease activation. An example is thrombin-antithrombin complex (TAT) which is a reporter of thrombin activation in vivo. TAT levels are elevated in thrombotic disease states and measurement of thrombin-antithrombin complexes is useful in the diagnosis of arterial and venous thrombotic diseases as well as a biomarker of disease and therapy. In general, serpin inhibition of proteolytic activities are measured by following the inhibition of cognate protease in vitro or in vivo.

Using the above examples, it is clear the availability of a novel cloned serpin provides opportunity for adjunct or replacement therapy, and are useful for the identification of serpin agonists, or stimulators (which might stimulate and/or bias serpin action), as well as, in the identification of serpin inhibitors. All of which might be therapeutically useful under different circumstances. The serpin of the present invention can also be used as a scaffold to tailor-make specific protease inhibitors and prevent angiogenesis. In addition detection of the serpin-cognate protease complex can be a useful diagnostic tool.

Polynucleotides and polypeptides corresponding to a portion of the full-length LSI-01 polypeptide of the present invention, in addition to its encoding polynucleotides, have been described by Baker et. al., International Publication Number WO 00/12708, gene UNQ692. Baker et al. did not appreciate the fact that their UNQ692 gene was not representative of the entire coding region, nor the fact that the UNQ692 translation product was deficient in the N-terminal domain. Baker et al., have termed their peptide fragment, encoded by their UNQ692 polynucleotide fragment, as PRO1337. Baker et al. teach the PRO1337 peptide as representing a homologue of a human thyroxin-binding globulin (TBG; Genbank Accession No. gi|4507377), and did not appreciate the fact that the protein shares higher identity to Arg/lys protease inhibitor serpins in domains essential for serpin activity and specificity, as opposed to the hormone-binding serpins.

The inventors of the present invention describe herein, the polynucleotides corresponding to the full-length LSI-01 gene and its encoded polypeptide. Also provided are polypeptide alignments illustrating the strong conservation of the LSI-01 polypeptide to known protease inhibitor serpins, and the dissimilarity of the protein in key amino acid domains to TBG. As inferred above, the dissimilarity is particularly evident in the ‘bait’ region, and the flexible loop region, which are essential for determining serpin function and specificity, as shown in FIG. 2. Based on this strong conservation, the inventors have ascribed the LSI-01 polypeptide as having at least some serpin protease inhibitory activities, particularly inhibition of proteases exhibiting Arg/lys specificity. Data is also provided illustrating the unique tissue expression profile of the LSI-01 polypeptide in lymphoid tissues, which has not been appreciated heretofore.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of LSI-01 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the LSI-01 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the LSI-01 protein having the amino acid sequence shown in FIGS. 1A-B (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone, LSI-01 (bac708.clone 24; AL132708_FL, BA-14448, protease 6; and/or protease inhibitor 6), deposited as ATCC Deposit Number PTA-2766 on Dec. 8, 2000.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of LSI-01 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the LSI-01 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

The invention further provides an isolated LSI-01 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The invention also provides a machine readable storage medium which comprises the structure coordinates of LSI-01, including all or any parts of the heparin binding region, the cleaved reactive loop binding region, and reactive loop region. Such storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises said regions or similarly shaped homologous regions.

The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. Such compounds are potential inhibitors of LSI-01 or its homologues.

The invention also provides novel classes of compounds, and pharmaceutical compositions thereof, that are useful as inhibitors of LSI-01 or its homologues.

The invention further relates to a method of identifying a compound that modulates the biological activity of LSI-01, comprising the steps of, (a) combining a candidate modulator compound with LSI-01 having the sequence set forth in one or more of SEQ ID NO:2; and measuring an effect of the candidate modulator compound on the activity of LSI-01.

The invention further relates to a method of identifying a compound that modulates the biological activity of a serpin, comprising the steps of, (a) combining a candidate modulator compound with a host cell expressing LSI-01 having the sequence as set forth in SEQ ID NO:2; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed LSI-01.

The invention further relates to a method of identifying a compound that modulates the biological activity of LSI-01, comprising the steps of, (a) combining a candidate modulator compound with a host cell containing a vector described herein, wherein LSI-01 is expressed by the cell; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed LSI-01.

The invention further relates to a method of screening for a compound that is capable of modulating the biological activity of LSI-01, comprising the steps of: (a) providing a host cell described herein; (b) determining the biological activity of LSI-01 in the absence of a modulator compound; (c) contacting the cell with the modulator compound; and (d) determining the biological activity of LSI-01 in the presence of the modulator compound; wherein a difference between the activity of LSI-01 in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.

The invention further relates to a compound that modulates the biological activity of human LSI-01 as identified by the methods described herein.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIGS. 1A-B show the polynucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of the novel human serpin, LSI-01, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 1766 nucleotides (SEQ ID NO:1), encoding a polypeptide of 435 amino acids (SEQ ID NO:2). An analysis of the LSI-01 polypeptide determined that it comprised the following features: a predicted signal peptide located from about amino acid 1 to about amino acid 38 of SEQ ID NO:2 (FIGS. 1A-B) represented by double underlining; a conserved serpin domain signature from about amino acid 405 to about amino acid 415 of SEQ ID NO:2 (FIGS. 1A-B) represented by shading; a flexible loop region characteristic of protease inhibitor serpins from about amino acid 384 to about amino acid 387 of SEQ ID NO:2 (FIGS. 1A-B) represented by single underlining; and a protease cleaveable bait region (also referred to as a ‘P1’ site) characteristic of trypsin-like cognate protease from about amino acid 395 to about amino acid 396 of SEQ ID NO:2 (FIGS. 1A-B) represented by bold amino acids.

FIG. 2 shows the regions of identity between the encoded LSI-01 protein (SEQ ID NO:2) to other serpins, specifically, the human α₁-antichymotrypsin protein (AACT_HUMAN; Genbank Accession No:gi|1 12874; Chandra et al., 1983; SEQ ID NO:3), the human Kallistatin protein, also known as Protease Inhibitor 4 (KAIN_HUMAN; Genbank Accession No:gi|5453888; Chai et al., 1993; SEQ ID NO:4), and the human thyroxin-binding globulin protein (THBG_HUMAN; Genbank Accession No:gi|37142; Flink et al., 1986; SEQ ID NO:5). The alignment was performed using the CLUSTALW algorithm described elsewhere herein. The shaded amino acids represent regions of matching identity. Lines between residues indicate gapped regions of non-identity for the aligned polypeptides. The black arrows above the alignment denote the characteristic signature peptide sequence of members of the SERPIN family of serine protease inhibitors (SEQ ID NO:21). The underlined (ATAA) sequence (SEQ ID NO:22) indicates the flexible region found in protease inhibitor serpins, which contrasts with the inflexible ATAV sequence of the thyroxin-binding globulin (Gils and Declerck, 1998).

FIG. 3 shows the regions of identity between the encoded LSI-01 protein (SEQ ID NO:2) to the human α₁-antithrypsin protein (pdb1qlp; Genbank Accession No:gi|6137432; SEQ ID NO:6). The alignment was performed using the CLUSTALW algorithm described elsewhere herein. The shaded amino acids represent regions of matching identity. Lines between residues indicate gapped regions of non-identity for the aligned polypeptides.

FIG. 4 shows a hydrophobicity plot of LSI-01 according to the BioPlot Hydrophobicity algorithm of Vector NTI (version 5.5). The hydrophilic peak at the N-terminus of the LSI-01 polypeptide is consistent with the presence of a predicted signal sequence.

FIG. 5 shows two expression profiles of the novel human serpin, LSI-01. The top panel illustrates the relative expression level of LSI-01 amongst various mRNA tissue sources. As shown, transcripts corresponding to LSI-01 expressed highly in the lymph node—more than 7-fold higher expression was observed in lymph node compared to the next highest level of tissue expression, the thymus. The bottom panel illustrates the same relative expression data as the top panel, though in the absence of the lymph node data, to more clearly show the proportional comparison between tissues of lower LSI-01 expression levels. As shown, the LSI-01 was expressed to a lesser extent, in the thymus, small intestine, and spleen. Expression data was obtained by measuring the steady state LSI-01 mRNA levels by quantitative PCR using the same PCR primer pair used to isolate the novel LSI-01 cDNA clone (SEQ ID NO:19 and 20) as described herein.

FIG. 6 shows a table illustrating the percent identity and percent similarity between the LSI-01 polypeptide of the present invention with other serpins, specifically, the human α₁-antichymotrypsin protein (AACT_HUMAN; Genbank Accession No:gi|112874; Chandra et al., 1983; SEQ ID NO:3), the human Kallistatin protein, also known as Protease Inhibitor 4 (KAIN_HUMAN; Genbank Accession No:gi|5453888; Chai et al., 1993; SEQ ID NO:4), the human thyroxin-binding globulin protein (THBG_HUMAN; Genbank Accession No:gi|37142; Flink et al., 1986; SEQ ID NO:5), and the human α₁-antithrypsin protein (pdb1qlp; Genbank Accession No:gi|6137432; SEQ ID NO:6). The percent identity and percent similarity values were determined using the BestFit algorithm using default parameters (Genetics Computer Group suite of programs).

FIG. 7 shows a three-dimensional homology model of the LSI-01 polypeptide based upon the homologous structure of the human α₁-antithrypsin protein (pdb1qlp; Genbank Accession No:; SEQ ID NO:6). The features of the human LSI-01 serpin identified in FIGS. 1A-B are labeled. Briefly, the predicted regions of beta sheet structure are represented in yellow; the predicted regions of alpha helix structure are represented in red; the predicted flexible loop region is represented in blue; the serpin bait P1 region is represented in red; and the consensus serpin region is presented in purple. Other regions of the protein structure are represented in aquamarine. The structural coordinates of the LSI-01 polypeptide are provided in Table III. The homology model of LSI-01 was derived from generating a sequence alignment with al anti-trypsin using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and the overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).

Table III provides the structural coordinates of the homology model of the LSI-01 polypeptide provided in FIG. 7 based upon the homologous structure of the human α₁-antithrypsin protein (pdb1qlp; Genbank Accession No:; SEQ ID NO:6). A description of the headings are as follows: “Atom” refers to the atom within the LSI-01 homology model; “Atom type” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid within which the atom resides; “#” refers to the amino acid number of the “residue”; “X”, “Y”, and “Z” structurally define the atomic position of the element measured in three dimensions; “B” is a thermal factor that measures movement of the atom around its atomic center; and “Occ” is an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates. A value of “1” indicates that each atom has the same conformation, i.e., the same position, in all molecules of the model.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein.

The invention provides a novel human sequence that encodes a serine protease inhibitor (serpin) with substantial homology to the class of serpins having a protease specificity for arginine/lysine residues (“trypsin-like” serine and similarly specific cysteine proteases). Proteases of this class of serpins include members of the blood coagulation cascade (clotting factors VIIa, Xa, IXa, thrombin), anticoagulation (protein C), fibrinolysis (tissue type plasminogen activator, urokinase and plasmin), complement pathways (complement factors C1 add others), proteases involved in inflammation (tryptase), and proteolytic digestion and signaling (trypsin). In addition, expression analysis indicates the LSI-01 has strong preferential expression in lymph nodes, and to a lesser extent, in thymus, small intestine, and spleen. Based on this information, we have provisionally named the gene and protein LSI-01 (Lymphocyte Specific Serine Protease Inhibitor-01.

In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or MRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.

In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:1 or the cDNA contained within the clone deposited with the ATCC. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without a signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ ID NO:1 was often generated by overlapping sequences contained in one or more clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:1 was deposited with the American Type Culture Collection (“ATCC”). As shown in Table I, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The deposited clone is inserted in the pSport1 plasmid (Life Technologies) using the NotI and SalI restriction endonuclease cleavage sites.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequence in FIGS. 1A-B (SEQ ID NO:1), a nucleic acid molecule of the present invention encoding the LSI-01 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 1A-B (SEQ ID NO:1) was discovered in a cDNA library derived from human liver and spleen.

The determined nucleotide sequence of the LSI-01 cDNA in FIGS. 1A-B (SEQ ID NO:1) contains an open reading frame encoding a protein of about 435 amino acid residues, with a deduced molecular weight of about 48.5 kDa. The amino acid sequence of the predicted LSI-01 polypeptide is shown in FIGS. 1A-B (SEQ ID NO:2). The LSI-01 protein shown in FIGS. 1A-B is about 46% identity and 52% similarity with the human α₁-antichymotrypsin protein (Genbank Accession No: gi|112874; SEQ ID NO:3); 48% identity and 56% similarity to the human Kallistatin (Genbank Accession No. gi|5453888; SEQ ID NO:4); 51% identity and 57% similarity to the human thyroxin-binding globulin (Genbank Accession No: gi|37142; SEQ ID NO:5); and 43% identity and 50% similarity to the human α₁-antithrypsin (Genbank Accession No: gi|6137432; SEQ ID NO:6), as shown in FIG. 6.

A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:1, the complement thereof, or the cDNA within the clone deposited with the ATCC. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6× SSPE (20× SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1× SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GP1 anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:1” refers to a polynucleotide sequence while “SEQ ID NO:2” refers to a polypeptide sequence, both sequences identified by an integer specified in Table I.

“A polypeptide having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)

The term “organism” as referred to herein is meant to encompass any organism referenced herein, though preferably to eukaryotic organsisms, more preferably to mammals, and most preferably to humans.

The present invention encompasses the identification of proteins, nucleic acids, or other molecules, that bind to polypeptides and polynucleotides of the present invention (for example, in a receptor-ligand interaction). The polynucleotides of the present invention can also be used in interaction trap assays (such as, for example, that described by Ozenberger and Young (Mol Endocrinol., 9(10):1321-9, (1995); and Ann. N. Y. Acad. Sci., 7;766:279-81, (1995)).

The polynucleotide and polypeptides of the present invention are useful as probes for the identification and isolation of full-length cDNAs and/or genomic DNA which correspond to the polynucleotides of the present invention, as probes to hybridize and discover novel, related DNA sequences, as probes for positional cloning of this or a related sequence, as probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides, as probes to quantify gene expression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present invention may comprise one, two, three, four, five, six, seven, eight, or more membrane domains.

Also, in preferred embodiments the present invention provides methods for further refining the biological function of the polynucleotides and/or polypeptides of the present invention.

Specifically, the invention provides methods for using the polynucleotides and polypeptides of the invention to identify orthologs, homologs, paralogs, variants, and/or allelic variants of the invention. Also provided are methods of using the polynucleotides and polypeptides of the invention to identify the entire coding region of the invention, non-coding regions of the invention, regulatory sequences of the invention, and secreted, mature, pro-, prepro-, forms of the invention (as applicable).

In preferred embodiments, the invention provides methods for identifying the glycosylation sites inherent in the polynucleotides and polypeptides of the invention, and the subsequent alteration, deletion, and/or addition of said sites for a number of desirable characteristics which include, but are not limited to, augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

In further preferred embodiments, methods are provided for evolving the polynucleotides and polypeptides of the present invention using molecular evolution techniques in an effort to create and identify novel variants with desired structural, functional, and/or physical characteristics.

The present invention further provides for other experimental methods and procedures currently available to derive functional assignments. These procedures include but are not limited to spotting of clones on arrays, micro-array technology, PCR based methods (e.g., quantitative PCR), anti-sense methodology, gene knockout experiments, and other procedures that could use sequence information from clones to build a primer or a hybrid partner.

As used herein the terms “modulate or modulates” refer to an increase or decrease in the amount, quality or effect of a particular activity, DNA, RNA, or protein.

Polynucleotides and Polvpeptides of the Invention

Features of the Polypeptide Encoded by Gene No:1

The polypeptide of this gene provided as SEQ ID NO:2 (FIGS. 1A-B), encoded by the polynucleotide sequence according to SEQ ID NO:1 (FIGS. 1A-B), and/or encoded by the polynucleotide contained within the deposited clone, LSI-01, has significant homology at the nucleotide and amino acid level to a number of serpins, which include, for example, the human α₁-antichymotrypsin protein (Genbank Accession No: gi|112874; SEQ ID NO:3); the human Kallistatin (Genbank Accession No. gi|5453888; SEQ ID NO:4); the human thyroxin-binding globulin (Genbank Accession No: gi|37142; SEQ ID NO:5); and the human α₁-antithrypsin (Genbank Accession No: gi|6137432; SEQ ID NO:6). An alignment of the LSI-01 polypeptide with these proteins is provided in FIG. 2.

The LSI-01 polypeptide was determined to have 46% identity and 52% similarity with the human α₁-antichymotrypsin protein (Genbank Accession No: gi|112874; SEQ ID NO:3); 48% identity and 56% similarity to the human Kallistatin (Genbank Accession No. gi|5453888; SEQ ID NO:4); 51% identity and 57% similarity to the human thyroxin-binding globulin (Genbank Accession No: gi|37142; SEQ ID NO:5); and 43% identity and 50% similarity to the human α₁-antithrypsin (Genbank Accession No: gi|6137432; SEQ ID NO:6).

The human α₁-antichymotrypsin protein (Genbank Accession No: gi|112874; SEQ ID NO:3) is a member of the serpin family that is known to inhibit neutrophil cathepsin g and mast cell chymase, both of which can convert angiotensin i to the active angiotensin II. The human α₁-antichymotrypsin protein is an extracellular protein synthesized in the liver having plasma tissue specificity. Like the related alpha-1-antitrypsin, its concentration increases in the acute phase of inflammation or infection. Deciencies in the α₁-antichymotrypsin protein can be a cause of chronic obstructive pulmonary disease (COPD) or of occlusive cerebrovascular disease. Morever, some reports have suggested that the α₁-antichymotrypsin protein also plays additional roles outside of its intrinsic serpin activity as it has been shown to also have DNA binding activity suggesting it may be a transcriptional modulator. Additional information relative to this protein may be found in reference to the following publications: Biochemistry 22 (22), 5055-5061 (1983); J. Biol. Chem.. 265 (2), 1199-1207 (1990); Biochim. Biophys. Acta 997 (1-2), 90-95 (1989); J. Biol. Chem.. 258 (21), 12749-12752 (1983); J. Mol. Biol. 218 (3), 595-606 (1991); Nat. Struct. Biol. 3 (10), 888-893 (1996); Biochemistry 37 (10), 3297-3304 (1998); Genomics 17 (3), 740-743 (1993); FEBS Lett. 304 (1), 66-68 (1992; which are hereby incorporated herein by reference in their entirety.

The human Kallistatin (Genbank Accession No. gi|5453888; SEQ ID NO:4) is a member of the serpin family which binds to tissue kallikrein and inhibits kallikrein's kininogenase and amidolytic activities. Kallistatin has a unique reactive site P1-P1′ of Phe-Ser and is expressed predominately in human liver. Additional information relative to this protein may be found in reference to the following publications: J Biol Chem 1993 Nov. 15;268(32):24498-505; and Genomics 1994 Sep. 15;23(2):370-8; which are hereby incorporated herein by reference.

The human α₁-antithrypsin (Genbank Accession No: gi|6137432; SEQ ID NO:6) is also a member of the serpin superfamily, and is often referred to as an archetypal member of the serpin superfamily. Members of the family show structural homology based on a dominant A beta-sheet and a mobile reactive centre loop. The structure of wild-type alpha 1-antitrypsin reveals a hydrophobic pocket between s2A and helices D and E that is filled on reactive loop insertion and the formation of biologically relevant loop-sheet polymers. This pocket may provide a target for rational drug design to prevent the formation of polymers and the associated plasma deficiency, liver cirrhosis and emphysema. Additional information relative to this protein may be found in reference to the following publication: J Mol Biol 1998 Jan. 23;275(3):419-25; which is hereby incorporated herein by reference.

The LSI-01 polypeptide was determined to comprise a signal sequence from about amino acid 1 to about amino acid 38 of SEQ ID NO:2 (FIGS. 1A-B) according to the SPScan computer algorithm (Genetics Computer Group suite of programs). Based upon the predicted signal peptide cleavage site, the mature LSI-01 polypeptide is expected to be from about amino acid 39 to about amino acid 435 of SEQ ID NO:2 (FIGS. 1A-B). As this determination was based upon the prediction from a computer algorithm, the exact physiological cleavage site may vary, as discussed more particularly herein. In this context, the term “about” should be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 more amino acids in either the N- or C-terminal direction of the above referenced polypeptide. Polynucleotides encoding these polypeptides are also provided.

In addition to the mature polypeptide above, the polynucleotides encoding the mature polypeptide are also encompassed by the present invention. Specifically, from about nucleotide position 182 to about nucleotide position 1372 of SEQ ID NO:1 (FIGS. 1A-B).

In confirmation of the strong homology to known serpins, the LSI-01 polypeptide was determined to have a conserved serine protease inhibitor domain. As discussed more particularly herein, serpins a group of structurally related, high molecular weight (400 to 500 amino acids), extracellular, irreversible serine protease inhibitors with a well defined structural-functional characteristic: a reactive region that acts as a ‘bait’ for an appropriate serine protease. This region is found in the C-terminal part of these proteins. Non limiting examples of proteins which are known to belong to the serpin family are the following: Alpha-1 protease inhibitor (alpha-1-antitrypsin, contrapsin); Alpha-1-antichymotrypsin; Antithrombin III; Alpha-2-antiplasmin; Heparin cofactor II; Complement C1 inhibitor; Plasminogen activator inhibitors 1 (PAI-1) and 2 (PAI-2); Glia derived nexin (GDN) (Protease nexin I); Protein C inhibitor; Rat hepatocytes SPI-1, SPI-2 and SPI-3 inhibitors; Human squamous cell carcinoma antigen (SCCA) which may act in the modulation of the host immune response against tumor cells; Leukocyte elastase inhibitor which, in contrast to other serpins, is an intracellular protein; Neuroserpin, a neuronal inhibitor of plasminogen activators; and plasmin, Cowpox virus crma, an inhibitor of the thiol protease interleukin-1B converting enzyme (ICE).

The consensus pattern for members of the serpin family is as follows: [LIVMFY]-x-[LIVMFYAC]-[DNQ]-[RKHQS]-[PST]-F-[LIVMFY]-[LIVMFYC]-x-[LIVMFAH], where ‘x’ represents any intervening amino acid. The consensus is centered on a well conserved Pro-Phe sequence which is found ten to fifteen residues on the C-terminal side of the reactive bond. In position 6 of the pattern, Pro is found in most serpins. However, the LSI-01 polypeptide of the present invention is an exception.

More information relating to serpins can be found elsewhere herein, or in reference to the following publications: Carrell R., Travis J., Trends Biochem. Sci. 10:20-24 (1985); Carrell R., Pemberton P. A., Boswell D. R., Cold Spring Harbor Symp. Quant. Biol. 52:527-535 (1987); Huber R., Carrell R. W., Biochemistry 28:8951-8966 (1989); Remold-O'Donneel E., FEBS Lett. 315:105-108 (1993); Osterwalder T., Contartese J., Stoeckli E. T., Kuhn T. B., Sonderegger P., EMBO J. 15:2944-2953 (1996); Komiyama T., Ray C. A., Pickup D. J., Howard A. D., Thornberry N. A., Peterson E. P., Salvesen G., J. Biol. Chem... 269:19331-19337 (1994); Clarke E., Sandwal B. D., Biochim. Biophys. Acta 1129:246-248 (1992); Zou Z., Anisowicz A., Neveu M., Rafidi K., Sheng S., Sager R., Hendrix M. J., Seftor E., Thor A., Science 263:526-529 (1994); Steele F. R., Chader G. J., Johnson L. V., Tombran-Tink J., Proc. Natl. Acad. Sci. U.S.A. 90:1526-1530 (1993); and Holland L. J., Suksang C., Wall A. A., Roberts L. R., Moser D. R., Bhattacharya A., J. Biol. Chem... 267:7053-7059 (1992).

In preferred embodiments, the following serpin domain polypeptides are encompassed by the present invention: VSFNRTFLMMI (SEQ ID NO:21), GTEATAATTTKFIVRS (SEQ ID NO:22), and/or PSYFTVSFNRTFLMMITNKAT (SEQ ID NO:23). Polynucleotides encoding these polypeptides are also provided.

In preferred embodiments, the LSI-01 polypeptide of the present invention is directed to a polypeptide having structural similarity to serpins. More preferred, is the LSI-ol polypeptide comprising a bait region with the amino acid, arginine (Arg), located in the P1 site. Based upon this structure, the LSI-01 polypeptide is expected to recognize proteases with Arg/lys specificity (“trypsin-like” serine and similarly specific cysteine proteases). As mentioned elsewhere herein, serpins inhibit multiple proteases with varying reactivity. The serpin of the present invention may inhibit several proteases both in vitro and in vivo.

Based upon the strong homology to members of the serine protease inhibitor (serpin) proteins, the LSI-01 polypeptide is expected to share at least some biological activity with serpins, and preferable with the arg/lys specific serpins, preferably the LSI-01 polypeptide shares at least some biological activity with the serpins disclosed herein, or otherwise known in the art.

Expression profiling designed to measure the steady state mRNA levels encoding the LSI-01 polypeptide showed predominately high expression levels in lymph node tissue, and to a lesser extent, in thymus, small intestine, and spleen tissue (See FIG. 5).

Moreover, the LSI-01 polynucleotides have been mapped to chromosome 14q32.1 (See Example 54). This region has been shown to contain genes encoding 6 additional serpins (A1-A6) and a protease Z inhibitor, and is commonly involved in several chromosomal aberrations including rearrangements, inversion, and/or translocations. Such aberrations within this region of chromosome 14 have been associated with the incidence of T-cell malignancy (T-cell neoplasms, leukemias; Saitou et al,. Identification of the TCL6 genes within the breakpoint cluster region on chromosome 14q32 in T-cell leukemia. Oncogene 19: 2796-2802, 2000.), or B-cell leukemias (i.e. one of the most frequent hematologic malignancies, follicular lymphoma is associated with a reciprocal translocation t(14;18)(q32;q21) and can result in deregulated expression of a chromosome 18 gene, Bcl2, which interferes with normal apoptosis of B lymphocytes; Ngan, B.-Y. et al. Expression in non-Hodgkin's lymphoma of the BCL-2 protein associated with the t(14;18) chromosomal translocation. New Eng. J. Med. 318:1638-1644, 1988). The oncogene activated by these different rearrangements is not always the same, and is dependent upon where the chromosomal breakpoints occur. Several candidate oncogenes responsible for malignant transformation have already been identified in chromosome 14q32 (i.e. TCL1A (T-cell leukemia/lymphoma 1a), TCL1b, TCL6, TRIP11 (Thyroid Hormone Receptor Interactor 11)).

Based on its expression profile and genetic chromosome map, the LS-1 polynucleotides could be a novel specific biomarker that would be useful in pinpointing subgroups of patients with chronic lymphocytic leukemias (with a distinct prognostic). Moreover, the LS-1 polynucleotides could also be useful as a diagnostic for identifying patients with chronic lymphocytic leukemias.

The LSI-01 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating serine protease activity in various cells, tissues, and organisms, and particularly in mammalian lymph node, thymus, small intestine, and spleen tissue, preferably human tissue. LSI-01 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing immune, gastrointestinal, metabolic, and/or proliferative diseases or disorders.

In preferred embodiments, LSI-01 polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following diseases or disorders: emphysema, liver cirrhosis, hepatocellular carcinoma, thrombosis, embolisms, thrombin-mediated vascular injury, microcirculation in sever sepsis, arterial thrombosis, myocardial infarction, unstable angina, stroke, venous thrombosis, and pulmonary embolism.

The strong homology to human serine protease inhibitors (serpins), combined with the predominate localized expression in lymph node tissue suggests the LSI-01 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing immune diseases and/or disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, and elsewhere herein. Briefly, the strong expression in immune tissue indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Such roles for serpins have been described. For example, the serpin described by Mueller et al., Eur-J-Immunol. 27(12): 3130-4 (1997) is believed to be involved to function as a megakaryocyte maturation factor in the presence of interleukin (IL)-3 and IL-11.

The LSI-01 polypeptide may also be useful as a preventative agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product may be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

Moreover, the protein would be useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

In addition, the strong homology to human serpins, combined with the expression in small intestine tissue emphasizes the potential utility for LSI-01 polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing gastrointestinal disorders, such as, for example, ulcers, cancers, etc., in addition to those disorders related to aberrant function of immune cells or tissue within the small intestine (e.g., Peyer's patches, etc.).

Moreover, the LSI-01 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing gastrointesinal diseases and/or disorders, which include, but are not limited to, ulcers, irritable bowel syndrome, inflammatory bowel disease, diarrhea, traveler's diarrhea, drug-related diarrhea polyps, absorption disorders, constipation, diverticulitis, vascular disease of the intestines, intestinal obstruction, intestinal infections, ulcerative colitis, Shigellosis, cholera, Crohn's Disease, amebiasis, enteric fever, Whipple's Disease, peritonitis, intrabdominal abcesses, hereditary hemochromatosis, gastroenteritis, viral gastroenteritis, food poisoning, mesenteric ischemia, mesenteric infarction, in addition to, metabolic diseases and/or disorders.

Moreover, polynucleotides and polypeptides, including fragments and/or antagonists thereof, have uses which include, directly or indirectly, treating, preventing, diagnosing, and/or prognosing susceptibility to the following, non-limiting, gastrointestinal infections: Salmonella infection, E.coli infection, E.coli 0157:H7 infection, Shiga Toxin-producing E.coli infection, Campylobacter infection (e.g., Campylobacter fetus, Campylobacter upsaliensis, Campylobacter hyointestinalis, Campylobacter lari, Campylobacter jejuni, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter rectus, Campylobacter curvus, Campylobacter sputorum, etc.), Heliobacter infection (e.g., Heliobacter cinaedi, Heliobacter fennelliae, etc.) Yersinia enterocolitica infection, Vibrio sp. Infection (e.g., Vibrio mnimicus, Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio vulnificus, Vibrio alginolyticus, Vibrio metschnikovii, Vibrio damsela, Vibrio cincinnatiensis, etc.) Aeromonas infection (e.g., Aeromonas hydrophila, Aeromonas sobira, Aeromonas caviae, etc.), Plesiomonas shigelliodes infection, Giardia infection (e.g., Giardia lamblia, etc.), Cryptosporidium infection, Listeria infection, Entamoeba histolytica infection, Rotavirus infection, Norwalk virus infection, Clostridium difficile infection, Clostriudium perfringens infection, Staphylococcus infection, Bacillus infection, in addition to any other gastrointestinal disease and/or disorder implicated by the causative agents listed above or elsewhere herein.

In addition, antagonists of the LSI-01 polynucleotides and polypeptides may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to hyper serine protease activity, which may include immune and/or proliferative diseases or disorders, particularly thrombosis, embolism, and other blood disorders. Therapeutic and/or pharmaceutical compositions comprising the LSI-01 polypeptides may be formulated to comprise heparin.

Moreover, LSI-01 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include treating, diagnosing, prognosing, and/or preventing hyperproliferative disorders, particularly of the immune and gastrointestinal systems. Such disorders may include, for example, cancers, and metastasis.

As the polypeptide of the present invention may be involved in the regulation of cytokine production, antigen presentation, T-cell maturation, or other processes, either directly or indirectly, suggests the LSI-01 polypeptide may be useful for the treatment of cancer (e.g. by boosting immune responses). A number of studies have shown a positive correlation between increased metastasis invasion to neighboring and systemic cells and tissues, to increased proteolytic activity of serine proteases (Testa et al., Cancer Metastasis Rev. 9:353, 1990; Dano et al., Adv. Cancer Res. 44:139, 1985; Foekens et al., Cancer Res. 52:6101, 1992; Ossowsky, Cancer Res. 52:6754, 1992; Sumiyoshi, Int. J. Cancer 50:345, 1992; Duffy et al., Cancer Res. 50:6827, 1992; and Meissauer et al., Exp. Cell Res. 192:453, 1991). The principal serine proteases known to be associated with tumor invasion mediate the plasminogen activation cascade. In this pathway, plasminogen is converted by plasminogen activators to plasmin, which is a wide-spectrum serine protease that degrades many components of the ECM directly, or indirectly via the activation of metalloproteases. The activity of the plasminogen activators is negatively regulated by plasminogen activator inhibitory proteins: PAI-1, PAI-2, and protease nexins (Chen, Current Opin. Cell Biol. 4:802, 1992).

The increased expression levels of uPA (urokinase plasminogen activator), the physiological substrate of alpha-anti-trypsin, has also been positively associated with increased incidence of cancers (Testa et al., Cancer Metastasis Rev. 9:353, 1990; Dano et al., Adv. Cancer Res. 44:139, 1985; Foekens et al., Cancer Res. 52:6101, 1992; Ossowsky, Cancer Res. 52:6754, 1992; Sumiyoshi, Int. J. Cancer 50:345, 1992; Duffy et al., Cancer Res. 50:6827, 1992; and Meissauer et al., Exp. Cell Res. 192:453, 1991; Heidtmann et al., Cancer Res. 49:6960, 1989; Sumiyoshi et al., Thromb Res. 63:59, 1991; Reilly et al., Int. J. Cancer 50:208, 1992, Cajot et al., Proc. Natl. Acad. Sci. USA 87:6939, 1990; Foucre et al., Br. J. Cancer 64:926, 1991; Shirasuna et al., Cancer Res. 53:147, 1993; and Janicke et al., Br. Can. Res. & Treat. 24:195, 1993).

The positive correlation between increased uPA expression on cancer incidence may have particular relevance to the LSI-01 polypeptide as the homology model of the present invention was based upon the structure of alpha-anti-trypsin, and may further suggest a use of the LSI-01 polypeptides, including fragments or antagonists thereof, in ameliorating or preventing metastasis, particularly in immune cells and tissues.

Moreover, based upon the chromosome mapping of LSI-01 to chromosome 14, the LSI-01 polynucleotides and polypeptides, including fragments and antagonists thereof, may have uses which include detecting, diagnosing, treating, ameliorating, and/or preventing leukemias, which include, are not limited to, T-cell malignancy (T-cell neoplasms, leukemias; Saitou et al,. Identification of the TCL6 genes within the breakpoint cluster region on chromosome 14q32 in T-cell leukemia. Oncogene 19: 2796-2802, 2000.), or B-cell leukemias (i.e. one of the most frequent hematologic malignancies, follicular lymphoma is associated with a reciprocal translocation t(14;18)(q32;q21) and can result in deregulated expression of a chromosome 18 gene, Bcl2, which interferes with normal apoptosis of B lymphocytes; Ngan, B.-Y. et al. Expression in non-Hodgkin's lymphoma of the BCL-2 protein associated with the t(14;18) chromosomal translocation. New Eng. J. Med. 318:1638-1644, 1988).

Despite the relatively specific expression in lymph node and other immune tissues, the LSI-01 polynucleotides and polypeptides, including fragments and antagonists thereof, may have uses which include detecting, diagnosing, treating, ameliorating, and/or preventing diseases and disorders of the neural system, particularly Alzheimer's disease, either directly or indirectly, in addition to other neural disorders known in the art or provided in the “Neurological Diseases” section herein. In support of this argument, Kalaria, R. N., et al. (Neurobiol-Aging,17(5): 687-93 (1996)) have reported that numerous molecular and cellular elements of the immune system are involved in the pathogenesis of Alzheimer's disease which include classical acute phase proteins of the pentraxin and serine protease inhibitor (serpin) families, as well as, a host of complement proteins and some coagulation factors.

Moreover, the LSI-01 polynucleotides and polypeptides, including fragments and /or antagonsists thereof, may have uses which include, directly or indirectly, diagnosing, prognosing, treating, preventing, and/or ameliorating cardiovascular, inflammatory diseases, particularly inflammatory diseases where proteases are known to be involved, certain cancers, diseases that result from uncontrolled complement fixation, diseases that result from uncontrolled blood coagulation, uncontrolled fibrinolysis, and uncontrolled bleeding.

The LSI-01 polynucleotides and polypeptides, including fragments and /or antagonsists thereof, may have uses which include identification of modulators of LSI-01 serpin function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains of the LSI-01 protein could be used as diagnostic agents of inflammatory conditions in patients, are useful in monitoring the activation and presence of cognate proteases, and can be used as a biomarker for the protease involvement in disease states and in the evaluation of inhibitors of the cognate protease in vivo.

Molecular genetic manipulation of the structure of the loop and of other functional domains in the serpin superfamily enables the production of serpins with tailor-made activities. Thus, the LSI-01 polypeptides, and fragments thereof, as well as any homologous product resulting from genetic manipulation of the structure, are useful for NMR-based design of modulators of LSI-01 biological activity, and serpins, in general.

LSI-01 polypeptides and polynucleotides have additional uses which include diagnosing diseases related to the over and/or under expression of LSI-01 by identifying mutations in the LSI-01 gene by using LSI-01 sequences as probes or by determining LSI-01 protein or mRNA expression levels. LSI-01 polypeptides may be useful for screening compounds that affect the activity of the protein. LSI-01 peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with LSI-01 (described elsewhere herein). Based on the expression pattern of this novel sequence, diseases that can be treated with agonists and/or antagonists for LSI-01 include various forms of generalized epilepsy.

Although it is believed the encoded polypeptide may share at least some biological activities with human serpin proteins (particularly serpins with Arg/Lys specificity), a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the LSI-01 polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from diseased immune tissue, as compared to, normal tissue might indicate a function in modulating immune function, for example. In the case of LSI-01, lymph node, thymus, small intestine, and/or spleen tissue should be used to extract RNA to prepare the probe.

In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the LSI-01 gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. In the case of LSI-01, a disease correlation related to LSI-01 may be made by comparing the MRNA expression level of LSI-01 in normal tissue, as compared to diseased tissue (particularly diseased tissue isolated from the following: lymph node, thymus, small intestine, and spleen tissue). Significantly higher or lower levels of LSI-01 expression in the diseased tissue may suggest LSI-01 plays a role in disease progression, and antagonists against LSI-01 polypeptides would be useful therapeutically in treating, preventing, and/or ameliorating the disease. Alternatively, significantly higher or lower levels of LSI-01 expression in the diseased tissue may suggest LSI-01 plays a defensive role against disease progression, and agonists of LSI-01 polypeptides may be useful therapeutically in treating, preventing, and/or ameliorating the disease. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:1 (FIGS. 1A-B).

The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the LSI-01, transforming yeast deficient in serpin activity, particularly Arg/Lys specificity serpin activity, and assessing their ability to grow would provide convincing evidence the LSI-01 polypeptide has serine protease inhibitor activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.

Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a lymph node, thymus, small intestine, or spleen specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.

In the case of LSI-01 transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (immune or gastrointestinal disorders, cancers, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal deletion mutants are encompassed by the present invention: M1-S435, Q2-S435, G3-S435, Q4-S435, G5-S435, R6-S435, R7-S435, R8-S435, G9-S435, TIO-S435, C11-S435, K12-S435, D13-S435, 114-S435, F15-S435, C16-S435, S17-S435, K18-S435, M19-S435, A20-S435, S21-S435, Y22-S435, L23-S435, Y24-S435, G25-S435, V26-S435, L27-S435, F28-S435, A29-S435, V30-S435, G31-S435, L32-S435, C33-S435, A34-S435, P35-S435, I36-S435, Y37-S435, C38-S435, V39-S435, S40-S435, P41-S435, A42-S435, N43-S435, A44-S435, P45-S435, S46-S435, A47-S435, Y48-S435, P49-S435, R50-S435, P51-S435, S52-S435, S53-S435, T54-S435, K55-S435, S56-S435, T57-S435, P58-S435, A59-S435, S60-S435, Q61-S435, V62-S435, Y63-S435, S64-S435, L65- S435, N66-S435, T67-S435, D68-S435, F69-S435, A70-S435, F71-S435, R72-S435, L73-S435, Y74-S435, R75-S435, R76-S435, L77-S435, V78-S435, L79-S435, E80-S435, T81-S435, P82-S435, S83-S435, Q84-S435, N85-S435, 186-S435, F87-S435, F88-S435, S89-S435, P90-S435, V91-S435, S92-S435, V93-S435, S94-S435, T95-S435, S96-S435, L97-S435, A98-S435, M99-S435, L1OO-S435, S101-S435, L102-S435, G103-S435, A104-S435, H105-S435, S106-S435, V107-S435, T108-S435, K109-S435, T110-S435, Q111-S435, I112-S435, L113-S435, Q114-S435, G115-S435, L116-S435, G117-S435, F118-S435, N119-S435, L120-S435, T121-S435, H122-S435, T123-S435, P124-S435, E125-S435, S126-S435, A127-S435, I128-S435, H129-S435, Q130-S435, G131-S435, F132-S435, Q133-S435, H134-S435, L135-S435, V136-S435, H137-S435, S138-S435, L139-S435, T140-S435, V141-S435, P142-S435, S143-S435, K144-S435, D145-S435, L146-S435, T147-S435, L148-S435, K149-S435, M150-S435, G151-S435, S152-S435, A153-S435, L154-S435, F155-S435, V156-S435, K157-S435, K158-S435, E159-S435, L160-S435, Q161-S435, L162-S435, Q163-S435, A164-S435, N165-S435, F166-S435, L167-S435, G168-S435, N169-S435, V170-S435, K171-S435, R172-S435, L173-S435, Y174-S435, E175-S435, A176-S435, E177-S435, V178-S435, F179-S435, S180-S435, T181-S435, D182-S435, F183-S435, S184-S435, N185-S435, P186-S435, S187-S435, 1188-S435, A189-S435, Q190-S435, A191-S435, R192-S435, I193-S435, N194-S435, S195-S435, H196-S435, V197-S435, K198-S435, K199-S435, K200-S435, T201-S435, Q202-S435, G203-S435, K204-S435, V205-S435, V206-S435, D207-S435, I208-S435, I209-S435, Q210-S435, G211-S435, L212-S435, D213-S435, L214-S435, L215-S435, T216-S435, A217-S435, M218-S435, V219-S435, L220-S435, V221-S435, N222-S435, H223-S435, I224-S435, F225-S435, F226-S435, K227-S435, A228-S435, K229-S435, W230-S435, E231-S435, K232-S435, P233-S435, F234-S435, H235-S435, L236-S435, E237-S435, Y238-S435, T239-S435, R240-S435, K241-S435, N242-S435, F243-S435, P244-S435, F245-S435, L246-S435, V247-S435, G248-S435, E249-S435, Q250-S435, V251-S435, T252-S435, V253-S435, Q254-S435, V255-S435, P256-S435, M257-S435, M258-S435, H259-S435, Q260-S435, K261-S435, E262-S435, Q263-S435, F264-S435, A265-S435, F266-S435, G267-S435, V268-S435, D269-S435, T270-S435, E271-S435, L272-S435, N273-S435, C274-S435, F275-S435, V276-S435, L277-S435, Q278-S435, M279-S435, D280-S435, Y281-S435, K282-S435, G283-S435, D284-S435, A285-S435, V286-S435, A287-S435, F288-S435, F289-S435, V290-S435, L291-S435, P292-S435, S293-S435, K294-S435, G295-S435, K296-S435, M297-S435, R298-S435, Q299-S435, L300-S435, E301-S435, Q302-S435, A303-S435, L304-S435, S305-S435, A306-S435, R307-S435, T308-S435, L309-S435, 1310-S435, K311-S435, W312-S435, S313-S435, H314-S435, S315-S435, L316-S435, Q317-S435, K318-S435, R319-S435, W320-S435, I321-S435, E322-S435, V323-S435, F324-S435, 1325-S435, P326-S435, R327-S435, F328-S435, S329-S435, I330-S435, S331-S435, A332-S435, S333-S435, Y334-S435, N335-S435, L336-S435, E337-S435, T338-S435, 1339-S435, L340-S435, P341-S435, K342-S435, M343-S435, G344-S435, I345-S435, Q346-S435, N347-S435, A348-S435, F349-S435, D350-S435, K351-S435, N352-S435, A353-S435, D354-S435, F355-S435, S356-S435, G357-S435, 1358-S435, A359-S435, K360-S435, R361-S435, D362-S435, S363-S435, L364-S435, Q365-S435, V366-S435, S367-S435, K368-S435, A369-S435, T370-S435, H371-S435, K372-S435, A373-S435, V374-S435, L375-S435, D376-S435, V377-S435, S378-S435, E379-S435, E380-S435, G381-S435, T382-S435, E383-S435, A384-S435, T385-S435, A386-S435, A387-S435, T388-S435, T389-S435, T390-S435, K391-S435, F392-S435, 1393-S435, V394-S435, R395-S435, S396-S435, K397-S435, D398-S435, G399-S435, P400-S435, S401-S435, Y402-S435, F403-S435, T404-S435, V405-S435, S406-S435, F407-S435, N408-S435, R409-S435, T410-S435, F411-S435, L412-S435, M413-S435, M414-S435, I415-S435, T416-S435, N417-S435, K418-S435, A419-S435, T420-S435, D421-S435, G422-S435, 1423-S435, L424-S435, F425-S435, L426-S435, G427-S435, K428-S435, and/or V429-S435 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these LSI-01 N-terminal deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal deletion mutants are encompassed by the present invention: M1-S435, M1-K434, M1-T433, M1-P432, M1-N431, M1-E430, M1-V429, M1-K428, M1-G427, M1-L426, M1-F425, M1-L424, M1-1423, M1-G422, M1-D421, M1-T420, M1-A419, M1-K418, M1-N417, M1-T416, M1-I415, M1-M414, M1-M413, M1-L412, M1-F411, M1-T410, M1-R409, M1-N408, M1-F407, M1-S406, M1-V405, M1-T404, M1-F403, M1-Y402, M1-S401, M1-P400, M1-G399, M1-D398, M1-K397, M1-S396, M1-R395, M1-V394, M1-1393, M1-F392, M1-K391, M1-T390, M1-T389, M1-T388, M1-A387, M1-A386, M1-T385, M1-A384, M1-E383, M1-T382, M1-G381, M1-E380, M1-E379, M1-S378, M1-V377, M1-D376, M1-L375, M1-V374, M1-A373, M1-K372, M1-H371, M1-T370, M1-A369, M1-K368, M1-S367, M1-V366, M1-Q365, M1-L364, M1-S363, M1-D362, M1-R361, M1-K360, M1-A359, M1-I358, M1-G357, M1-S356, M1-F355, M1-D354, M1-A353, M1-N352, M1-K351, M1-D350, M1-F349, M1-A348, M1-N347, M1-Q346, M1-1345, M1-G344, M1-M343, M1-K342, M1-P341, M1-L340, M1-1339, M1-T338, M1-E337, M1-L336, M1-N335, M1-Y334, M1-S333, M1-A332, M1-S331, M1-1330, M1-S329, M1-F328, M1-R327, M1-P326, M1-1325, M1-F324, M1-V323, M1-E322, M1-1321, M1-W320, M1-R319, M1-K318, M1-Q317, M1-L316, M1-S315, M1-H314, M1-S313, M1-W312, M1-K311, M1-I310, M1-L309, M1-T308, M1-R307, M1-A306, M1-S305, M1-L304, M1-A303, M1-Q302, M1-E301, M1-L300, M1-Q299, M1-R298, M1-M297, M1-K296, M1-G295, M1-K294, M1-S293, M1-P292, M1-L291, M1-V290, M1-F289, M1-F288, M1-A287, M1-V286, M1-A285, M1-D284, M1-G283, M1-K282, M1-Y281, M1-D280, M1-M279, Ml-Q278, M1-L277, M1-V276, M1-F275, M1-C274, M1-N273, M1-L272, M1-E271, M1-T270, M1-D269, M1-V268, M1-G267, M1-F266, M1-A265, M1-F264, M1-Q263, M1-E262, M1-K261, M1-Q260, M1-H259, M1-M258, M1-M257, M1-P256, M1-V255, M1-Q254, M1-V253, M1-T252, M1-V251, M1-Q250, M1-E249, M1-G248, M1-V247, M1-L246, M1-F245, M1-P244, M1-F243, M1-N242, M1-K241, M1-R240, M1-T239, M1-Y238, M1-E237, M1-L236, M1-H235, M1-F234, M1-P233, M1-K232, M1-E231, M1-W230, M1-K229, M1-A228, M1-K227, M1-F226, M1-F225, M1-1224, M1-H223, M1-N222, M1-V221, M1-L220, M1-V219, M1-M218, M1-A217, M1-T216, M1-L215, M1-L214, M1-D213, M1-L212, M1-G211, M1-Q210, M1-1209, M1-1208, M1-D207, M1-V206, M1-V205, M1-K204, M1-G203, M1-Q202, M1-T201, M1-K200, M1-K199, M1-K198, M1-V197, M1-H196, M1-S195, M1-N194, M1-1193, M1-R192, M1-A191, M1-Q190, M1-A189, M1-I188, M1-S187, M1-P186, M1-N185, M1-S184, M1-F183, M1-D182, M1-T181, M1-S180, M1-F179, M1-V178, M1-E177, M1-A176, M1-E175, M1-Y174, M1-L173, M1-R172, M1-K171, M1-V170, M1-N169, M1-G168, M1-L167, M1-F166, M1-N165, M1-A164, M1-Q163, M1-L162, M1-Q161, M1-L160, M1-E159, M1-K158, M1-K157, M1-V156, M1-F155, M1-L154, M1-A153, M1-S152, M1-G151, M1-M150, M1-K149, M1-L148, M1-T147, M1-L146, M1-D145, M1-K144, M1-S143, M1-P142, M1-V141, M1-T140, M1-L139, M1-S138, M1-H137, M1-V136, M1-L135, M1-H134, M1-Q133, M1-F132, M1-G131, M1-Qi30, M1-H129, M1-I128, M1-A127, M1-S126, M1-E125, M1-P124, M1-T123, M1-H122, M1-T121, M1-L120, M1-N119, M1-F118, M1-G117, M1-L116, M1-G115, M1-Q114, M1-L113, M1-I112, M1-Q111, M1-T111, M1-K109, M1-T108, M1-V107, M1-S106, M1-H105, M1-A104, M1-G103, M1-L102, M1-S101, M1-L100, M1-M99, M1-A98, M1-L97, M1-S96, M1-T95, M1-S94, M1-V93, M1-S92, M1-V91, M1-P90, M1-S89, M1-F88, M1-F87, M1-I86, M1-N85, M1-Q84, M1-S83, M1-P82, M1-T81, M1-E80, M1-L79, M1-V78, M1-L77, M1-R76, M1-R75, M1-Y74, M1-L73, M1-R72, M1-F71, M1-A70, M1-F69, M1-D68, M1-T67, M1-N66, M1-L65, M1-S64, M1-Y63, M1-V62, M1-Q61, M1-S60, M1-A59, M1-P58, M1-T57, M1-S56, M1-K55, M1-T54, M1-S53, M1-S52, M1-P51, M1-R50, M1-P49, M1-Y48, M1-A47, M1-S46, M1-P45, M1-A44, M1-N43, M1-A42, M1-P41, M1-S40, M1-V39, M1-C38, M1-Y37, M1-I36, M1-P35, M1-A34, M1-C33, M1-L32, M1-G31, M1-V30, M1-A29, M1-F28, M1-L27, M1-V26, M1-G25, M1-Y24, M1-L23, M1-Y22, M1-S21, Mi-A20, M1-M19, M1-K18, M1-S17, M1-C16, M1-F15, M1-I14, M1-D13, M1-K12, M1-C11, M1-T10, M1-G9, M1-R8, and/or M1-R7 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these LSI-01 C-terminal deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenic epitopes of the LSI-01 polypeptide.

In preferred embodiments, the following immunogenic and/or antigenic epitope polypeptides are encompassed by the present invention: amino acid residues from about amino acid 133 to about amino acid 146, from about amino acid 133 to about amino acid 141, from about amino acid 138 to about amino acid 146, from about amino acid 155 to about amino acid 163, from about amino acid 193 to about amino acid 204, from about amino acid 193 to about amino acid 201, from about amino acid 196 to about amino acid 204, from about amino acid 228 to about amino acid 240, from about amino acid 228 to about amino acid 236, from about amino acid 232 to about amino acid 240, from about amino acid 302 to about amino acid 318, from about amino acid 302 to about amino acid 310, from about amino acid 310 to about amino acid 318, from about 361 to about 377, from about 361 to about 369, and/or from about 369 to about 377 of SEQ ID NO:2 (FIGS. 1A-B). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-terminus and/or C-terminus of the above referenced polypeptide. Polynucleotides encoding this polypeptide are also provided.

The LSI-01 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the LSI-01 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the LSI-01 polypeptide to associate with other polypeptides, particularly the serine protease substrate for LSI-01, or its ability to modulate serine protease function.

Specifically, the LSI-01 polypeptide was predicted to comprise one tyrosine phosphorylation site using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or [RK]-x(3)-[DE]-x(2)-Y, where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973-977(1982); Hunter T., J. Biol. Chem... 257:4843-4848(1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem... 259:7835-7841(1984), which are hereby incorporated herein by reference.

In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: TKFIVRSKDGPSYFTVSF (SEQ ID NO:17). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LSI-01 tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The LSI-01 polypeptide was predicted to comprise eight PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem... 260:12492-12499(1985); which are hereby incorporated by reference herein.

In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: GRRRGTCKDIFCS (SEQ ID NO:9), YPRPSSTKSTPAS (SEQ ID NO:10), PSKDLTLKMGSAL (SEQ ID NO:11), FHLEYTRKNFPFL (SEQ ID NO:12), LEQALSARTLIKW (SEQ ID NO:13), QVSKATHKAVLDV (SEQ ID NO:14), ATAATTTKFIVRS (SEQ ID NO:15), and/or FLMMITNKATDGI (SEQ ID NO:16). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LSI-01 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The LSI-01 polypeptide was predicted to comprise one casein kinase II phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). Casein kinase II (CK-2) is a protein serine/threonine kinase whose activity is independent of cyclic nucleotides and calcium. CK-2 phosphorylates many different proteins. The substrate specificity [1] of this enzyme can be summarized as follows: (1) Under comparable conditions Ser is favored over Thr.; (2) An acidic residue (either Asp or Glu) must be present three residues from the C-terminal of the phosphate acceptor site; (3) Additional acidic residues in positions +1, +2, +4, and +5 increase the phosphorylation rate. Most physiological substrates have at least one acidic residue in these positions; (4) Asp is preferred to Glu as the provider of acidic determinants; and (5) A basic residue at the N-terminal of the acceptor site decreases the phosphorylation rate, while an acidic one will increase it.

A consensus pattern for casein kinase II phosphorylations site is as follows: [ST]-x(2)-[DE], wherein ‘x’ represents any amino acid, and S or T is the phosphorylation site.

Additional information specific to casein kinase II phosphorylation sites may be found in reference to the following publication: Pinna L. A., Biochim. Biophys. Acta 1054:267-284(1990); which is hereby incorporated herein in its entirety.

In preferred embodiments, the following casein kinase II phosphorylation site polypeptide is encompassed by the present invention: GRRRGTCKDIFCSK (SEQ ID NO:46). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this casein kinase II phosphorylation site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The LSI-01 polypeptide was predicted to comprise three cAMP- and cGMP-dependent protein kinase phosphorylation site using the Motif algorithm (Genetics Computer Group, Inc.). There has been a number of studies relative to the specificity of cAMP- and cGMP-dependent protein kinases. Both types of kinases appear to share a preference for the phosphorylation of serine or threonine residues found close to at least two consecutive N-terminal basic residues.

A consensus pattern for cAMP- and cGMP-dependent protein kinase phosphorylation sites is as follows: [RK](2)-x-[ST], wherein “x” represents any amino acid, and S or T is the phosphorylation site.

Additional information specific to cAMP- and cGMP-dependent protein kinase phosphorylation sites may be found in reference to the following publication: Fremisco J. R., Glass D. B., Krebs E. G, J. Biol. Chem.. 255:4240-4245(1980); Glass D. B., Smith S. B., J. Biol. Chem.. 258:14797-14803(1983); and Glass D. B., El-Maghrabi M. R., Pilkis S. J., J. Biol. Chem.. 261:2987-2993(1986); which is hereby incorporated herein in its entirety.

In preferred embodiments, the following cAMP- and cGMP-dependent protein kinase phosphorylation sites polypeptide are encompassed by the present invention: QGQGRRRGTCKDIF (SEQ ID NO:39), INSHVKKKTQGKVV (SEQ ID NO:40), and/or FSGIAKRDSLQVSK (SEQ ID NO:41). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these cAMP- and cGMP-dependent protein kinase phosphorylation site polypeptide as immunogenic and/or antigenic epitopes as described elsewhere herein.

The LSI-01 polypeptide has been shown to comprise two glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

In preferred embodiments, the following asparagine glycosylation site polypeptides are encompassed by the present invention: QGLGFNLTHTPESA (SEQ ID NO:7), and/or FTVSFNRTFLMMIT (SEQ ID NO:8). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LSI-01 asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The LSI-01 polypeptide has been shown to comprise one amidation site according to the Motif algorithm (Genetics Computer Group, Inc.). The precursor of hormones and other active peptides which are C-terminally amidated is always directly followed by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Val or Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. A consensus pattern for amidation sites is the following: x-G-[RK]-[RK], wherein “X” represents the amidation site. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F., Smyth D. G., Biosci. Rep. 7:907-916(1987).

In preferred embodiments, the following amidation site polypeptide is encompassed by the present invention: MQGQGRRRGTCK (SEQ ID NO:38). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this LSI-01 amidation site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The LSI-01 polypeptide was predicted to comprise four N-myristoylation sites using the Motif algorithm (Genetics Computer Group, Inc.). An appreciable number of eukaryotic proteins are acylated by the covalent addition of myristate (a C14-saturated fatty acid) to their N-terminal residue via an amide linkage. The sequence specificity of the enzyme responsible for this modification, myristoyl CoA:protein N-myristoyl transferase (NMT), has been derived from the sequence of known N-myristoylated proteins and from studies using synthetic peptides. The specificity seems to be the following: i.) The N-terminal residue must be glycine; ii.) In position 2, uncharged residues are allowed; iii.) Charged residues, proline and large hydrophobic residues are not allowed; iv.) In positions 3 and 4, most, if not all, residues are allowed; v.) In position 5, small uncharged residues are allowed (Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) In position 6, proline is not allowed.

A consensus pattern for N-myristoylation is as follows: G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein ‘x’ represents any amino acid, and G is the N-myristoylation site.

Additional information specific to N-myristoylation sites may be found in reference to the following publication: Towler D. A., Gordon J. I., Adams S. P., Glaser L., Annu. Rev. Biochem. 57:69-99(1988); and Grand R. J. A., Biochem. J. 258:625-638(1989); which is hereby incorporated herein in its entirety.

In preferred embodiments, the following N-myristoylation site polypeptides are encompassed by the present invention: ASYLYGVLFAVGLCAP (SEQ ID NO:42), TQILQGLGFNLTHTPE (SEQ ID NO:43), ELPKMGIQNAFDKNAD (SEQ ID NO:44), and/or DVSEEGTEATAATTTK (SEQ ID NO:45). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these N-myristoylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also provides a three-dimensional homology model of the LSI-01 polypeptide (see FIG. 7). The homology model of the LSI-01 polypeptide is based upon the homologous structure of the human α₁-antithrypsin protein (pdb1qlp; Genbank Accession No:; SEQ ID NO:6) and is defined by the set of structural coordinates set forth in Table III herein. A description of the headings in Table im are as follows: “Atom” refers to the atom within the LSI-01 homology model; “Atom type” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid within which the atom resides; “#” refers to the amino acid number of the “residue”; “X”, “Y”, and “Z” structurally define the atomic position of the element measured in three dimensions; “B” is a thermal factor that measures movement of the atom around its atomic center; and “Occ” is an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates. A value of “1” indicates that each atom has the same conformation, i.e., the same position, in all molecules of the model.

The LSI-01 homology model of the present invention may provide one basis for designing rational stimulators (agonists) and/or inhibitors (antagonists) of one or more of the biological functions of LSI-01, or of LSI-01 mutants having altered specificity (e.g., molecularly evolved LSI-01 polypeptides, engineered site-specific LSI-01 mutants, LSI-01 allelic variants, etc.).

Homology models are not only useful for designing rational agonists and/or antagonists, but are also useful in predicting the function of a particular polypeptide. The functional predictions from homology models are typically more accurate than the functional attributes derived from traditional polypeptide sequence homology alignments (e.g., CLUSTALW), particularly when the three dimensional structure of a related polypeptide is known (e.g., human α₁-antithrypsin protein). The increased prediction accuracy is based upon the fact that homology models approximate the three-dimensional structure of a protein, while homology based alignments only take into account the one dimension polypeptide sequence. Since the function of a particular polypeptide is determined not only by its primary, secondary, and tertiary structure, functional assignments derived solely upon homology alignments using the one dimensional protein sequence may be less reliable. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995).

Prior to developing a homology model, those of skill in the art would appreciate that a template of a known protein, or model protein, must first be identified which will be used as a basis for constructing the homology model for the protein of unknown structure (query template). In the case of the LSI-01 polypeptide of the present invention, the model protein template used in constructing the LSI-01 homology model was the human α₁-antithrypsin protein.

Identifying a template can be accomplished using pairwise alignment of protein sequences using such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul, et al, 1990). In cases where sequence similarity is high (greater than 30%), such pairwise comparison methods may be adequate for identifying an appropriate template. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques may be used. Such techniques, include, for example, protein fold recognition (protein threading; Hendlich, et al, 1990), where the compatibility of a particular polypeptide sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential.

Following the initial sequence alignment, the second step would be to optimally align the query template to the model template by manual manipulation and/or by the incorporation of features specific to the polypeptides (e.g., motifs, secondary structure predictions, and allowed conservations). Preferably, the incorporated features are found within both the model and query template.

The third step would be to identify structurally conserved regions that could be used to construct secondary core structure (Sali, et al, 1995). Loops could be added using knowledge-based techniques, and by performing forcefield calculations (Sali, et al, 1995).

The term “structure coordinates” refers to Cartesian coordinates generated from the three dimensional structure of a homology model. As referenced above, the homology model of the LSI-01 polypeptide was derived by first, generating a sequence alignment with al anti-trypsin using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and, secondly, generating an overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).

The skilled artisan would appreciate that a set of structure coordinates for a protein represents a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from the generation of similar homology models using different alignment templates (i.e., other than the human alphα₁-antitrypsin), and/or using different methods in generating the homology model, will likely have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table m could be manipulated by fractionalization of the structure coordinates; integer additions, or integer subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

Therefore, various computational analyses are necessary to determine whether a template molecule or a portion thereof is sufficiently similar to all or part of a query template in order to be considered the same. Such analyses may be carried out using software applications available in the art, such as, for example, INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000, as described in the accompanying User's Guide.

Using the superimposition tool in the program INSIGHTII, comparisons can be made between different structures and different conformations of the same structure. The procedure used in INSIGHTII to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. The atom equivalency within INSIGHTII is defined by user input. For the purpose of this invention, we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by the INSIGHTII program. For the purpose of the present invention, any homology model of a LSI-01 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table III are considered identical. More preferably, the root mean square deviation for the LSI-01 polypeptide is less than 2.0 Å.

The homology model of the present invention is useful for the structure-based design of modulators of the LSI-01 biological function, as well as mutants with altered biological function and/or specificity.

In accordance with the structural coordinates provided in Table III and the three dimensional homology model of LSI-01, the LSI-01 polypeptide has been shown to comprise a heparin binding region embodied by the following amino acids: from about amino acid Y63 to about amino acid E80, from about amino acid E125 to about amino acid T140, and/or from about amino acid A306 to about amino acid S315 of SEQ ID NO:2 (FIGS. 1A-B). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced polypeptides.

In preferred embodiments, the following LSI-01 heparin binding region polypeptides are encompassed by the present invention: YSLNTDFAFRLYRRLVLE (SEQ ID NO:24), ESAIHQGFQHLVHSLT (SEQ ID NO:25), and/or ARTLIKWSHS (SEQ ID NO:26). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LSI-01 heparin binding polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In accordance with the structural coordinates provided in Table III and the three dimensional homology model of LSI-01, the LSI-01 polypeptide has been shown to comprise a cleaved reactive loop binding region embodied by the following amino acids: from about amino acid N185 to about amino acid T201, from about amino acid Q202 to about amino acid 1209, from about amino acid A217 to about amino acid K227, and/or from about amino acid K368 to about amino acid V377 of SEQ ID NO:2 (FIGS. 1A-B). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced polypeptides.

In preferred embodiments, the following LSI-01 cleaved reactive loop binding region polypeptides are encompassed by the present invention: NPSIAQARINSHVKKKT (SEQ ID NO:27), QGKVVDII (SEQ ID NO:28), AMVLVNHIFFK (SEQ ID NO:29), and/or KATHKAVLDV (SEQ ID NO:30). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LSI-01 cleaved reactive loop binding region polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In accordance with the structural coordinates provided in Table III and the three dimensional homology model of LSI-01, the LSI-01 polypeptide has been shown to comprise a reactive loop region specified by the following amino acids: A384, T385, A386, A387, T388, T389, T390, K391, F392, 1393, V394, R395, S396, K397, D398, G399, P400, S401, Y402, F403, T404, and/or from about amino acid A384 to about amino acid T404 of SEQ ID NO:2 (FIGS. 1A-B). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced polypeptides.

In preferred embodiments, the following LSI-01 reactive loop region polypeptide is encompassed by the present invention: ATAATTTKFIVRSKDGPSYFT (SEQ ID NO:31). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the LSI-01 reactive loop region polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at least a portion of the LSI-01 reactive loop region (SEQ ID NO:31). Such polypeptides may correspond, for example, to the N- and/or C- terminal deletions of the reactive loop region.

In preferred embodiments, the following N-terminal reactive loop region deletions are encompassed by the present invention: A1-T21, T2-T21, A3-T21, A4-T21, T5-T21, T6-T21, T7-T21, K8-T21, F9-T21, I10-T21, V11-T21, R12-T21, S13-T21, K14-T21, and/or D15-T21 of SEQ ID NO:32. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following C-terminal reactive loop region deletions are encompassed by the present invention: A1-T21, A1-F20, A1-Y19, A1-S18, A1-P17, A1-G16, A1-D15, A1-K14, A1-S13, A1-R12, A1-V11, A1-I10, A1-F9, A1-K8, and/or A1-T7 of SEQ ID NO:32. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the reactive loop region (e.g., any combination of both N- and C- terminal reactive loop region deletions) of SEQ ID NO:32. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the reactive loop region (SEQ ID NO:31), and where CX refers to any C-terminal amino acid position of the reactive loop region (SEQ ID NO:31). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at least a portion of the LSI-01 heparin binding region (SEQ ID NO:24, 25, and/or 26). Such polypeptides may correspond, for example, to N- and/or C- terminal deletions of any one of the heparin binding regions.

In preferred embodiments, the following N-terminal heparin binding region deletions are encompassed by the present invention: Y1-E18, S2-E18, L3-E18, N4-E18, T5-E18, D6-E18, F7-E18, A8-E18, F9-E18, R10-E18, L11-E18, and/or Y12-E18 of SEQ ID NO:25. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following C-terminal heparin binding region deletions are encompassed by the present invention: Y1-E18, Y1-L17, Y1-V16, Y1-L15, Y1-R14, Y1-R13, Y1-Y12, Y1-L11, Y1-R10, Y1-F9, Y1-A8, and/or Y1-F7 of SEQ ID NO:25. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following N-terminal heparin binding region deletions are encompassed by the present invention: E1-T16, S2-T16, A3-T16, I4-T16, H5-T16, Q6-T16, G7-T16, F8-T16, Q9-T16, and/or H10-T16 of SEQ ID NO:26. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following C-terminal heparin binding region deletions are encompassed by the present invention: E1-T16, E1-L15, E1-S14, E1-H13, E1-V12, E1-L11, E1-H10, E1-Q9, E1-F8, and/or E1-G7 of SEQ ID NO:26. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following N-terminal heparin binding region deletions are encompassed by the present invention: A1-S10, R2-S10, T3-S10, and/or L4-S 10 of SEQ ID NO:27. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following C-terminal heparin binding region deletions are encompassed by the present invention: A1-S10, A1-H9, A1-S8, and/or A1-W7 of SEQ ID NO:27. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of any one of the heparin binding region polypeptides, such as any combination of both N- and C-terminal deletions for each respective region corresponding to SEQ ID NO:24, 25, or 26,. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of any one of the heparin binding regions (SEQ ID NO:24, 25, or 26), and where CX refers to any C-terminal amino acid position of any one of the heparin binding regions (SEQ ID NO:24, 25, or 26). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at least a portion of one of the LSI-01 cleaved reactive loop binding region polypeptides (SEQ ID NO:27, 28, 29, or 30). Such polypeptides may correspond, for example, to N- and/or C-terminal deletions of any one of the cleaved reactive loop binding region polypeptides.

In preferred embodiments, the following N-terminal cleaved reactive loop binding region deletions are encompassed by the present invention: N1-T17, P2-T17, S3-T17, I4-T17, A5-T17, Q6-T17, A7-T17, R8-T17, I9-T17, N10-T17, and/or S11-T17 of SEQ ID NO:27. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following C-terminal cleaved reactive loop binding region deletions are encompassed by the present invention: N1-T17, N1-K16, N1-K15, N1-K14, N1-V13, N1-H12, N1-S11, N1-N10, N1-I9, N1-R8, and/or N1-A7 of SEQ ID NO:27. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following N-terminal cleaved reactive loop binding region deletions are encompassed by the present invention: Q1-I8, and/or G2-I8 of SEQ ID NO:28. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following C-terminal cleaved reactive loop binding region deletions are encompassed by the present invention: Q1-18, and/or Q1-I7 of SEQ ID NO:28. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following N-terminal cleaved reactive loop binding region deletions are encompassed by the present invention: A1-K11, M2-K11, V3-K11, L4-K11, and/or V5-K11 of SEQ ID NO:29. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following C-terminal cleaved reactive loop binding region deletions are encompassed by the present invention: A1-K11, A1-F10, A1-F9, A1-I8, and/or A1-H7 of SEQ ID NO:29. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following N-terminal cleaved reactive loop binding region deletions are encompassed by the present invention: K1-V10, A2-V10, T3-V10, and/or H4-V10 of SEQ ID NO:30. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following C-terminal cleaved reactive loop binding region deletions are encompassed by the present invention: K1-V10, K1-D9, K1-L8, and/or K1-V7 of SEQ ID NO:30. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of any one of the cleaved reactive loop binding region polypeptides, such as any combination of both N- and C-terminal deletions for each respective region corresponding to SEQ ID NO:27, 28, 29, or 30. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of any one of the heparin binding regions (SEQ ID NO:27, 28, 29, or 30), and where CX refers to any C-terminal amino acid position of any one of the heparin binding regions (SEQ ID NO:27, 28, 29, or 30). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

For purposes of the present invention, by “at least a portion of” is meant all or any part of the reactive loop region, the heparin binding regions, and the cleaved reactive loop binding region defined by the structure coordinates according to Table III (e.g., fragments thereof). More preferred are molecules comprising all or any parts of the reactive loop region, the heparin binding regions, and the cleaved reactive loop binding region, according to Table ImI, or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said LSI-01 amino acids of not more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a term that expresses the deviation or variation from a trend or object. For the purposes of the present invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of the AR portion of the complex as defined by the structure coordinates described herein.

A preferred embodiment is a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structure coordinates of all of the amino acids in Table III ± a root mean square deviation from the backbone atoms of those amino acids of not more than 4.0 ANG, preferably 3.0 ANG.

The structure coordinates of a LSI-01 homology model, including portions thereof, is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table III.

One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.

Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.

In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.

For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.

Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the LSI-01 homology model, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.

For the first time, the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities that are capable of modulating the biological function of LSI-01.

Accordingly, the present invention is also directed to the design of small molecules which imitates the structure of the LSI-01 reactive loop region (SEQ ID NO:31), or a portion thereof, in accordance with the structure coordinates provided in Table III. Alternatively, the present invention is directed to the design of small molecules which may bind to at least part of the reactive loop region (SEQ ID NO:31), or some portion thereof. For purposes of this invention, by LSI-01 reactive loop, it is also meant to include mutants or homologues thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to SEQ ID NO:31. In this context, the term “small molecule” may be construed to mean any molecule described known in the art or described elsewhere herein, though may include, for example, peptides, chemicals, carbohydrates, nucleic acids, PNAs, and any derivatives thereof.

Accordingly, the present invention is also directed to the design of small molecules which imitates the structure of the cleaved reactive loop binding region polypeptides of LSI-01 (SEQ ID NO:27, 28, 29, or 30), or a portion thereof, in accordance with the structural coordinates provided in Table III. Alternatively, the present invention is directed to the design of small molecules which may bind to at least part of one of the cleaved reactive loop binding region polypeptides, or some portion thereof. For purposes of this invention, by LSI-01 cleaved reactive loop binding region, it is also meant to include mutants or homologues thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to SEQ ID NO:27, 28, 29, or 30 according to Table III. In this context, the term “small molecule” may be construed to mean any molecule described known in the art or described elsewhere herein, though may include, for example, peptides, chemicals, carbohydrates, nucleic acids, PNAs, and any derivatives thereof.

The present invention is also directed to the design of small molecules which imitates the structure of one of the heparin binding region polypeptides in LSI-01 (SEQ ID NO:24, 25, or 26), or some portion thereof, in accordance with the structure coordinates provided in Table III. Alternatively, the present invention is directed to the design of small molecules which may bind to at least part of one of the heparin binding region polypeptides, or some portion thereof. For the purposes of this invention, by LSI-01 heparin binding region, it is also meant to include mutants or homologues thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to (SEQ ID NO:24, 25, or 26) in accordance with the structure coordinates of Table III. In this context, the term “small molecule” may be construed to mean any molecule described known in the art or described elsewhere herein, though may include, for example, peptides, chemicals, carbohydrates, nucleic acids, PNAs, and any derivatives thereof.

The three-dimensional model structure of the LSI-01 will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.

For example, test compounds can be modeled that fit spatially into the heparin binding region in LSI-01 embodied by the sequence from about Y63 to about E80, from about E125 to about T140, from about A306 to about S315, or some portion thereof, of SEQ ID NO:2 (corresponding to SEQ ID NO:24, 25, or 26,, respectively), in accordance with the structural coordinates of Table III.

For example, test compounds can be modeled that fit spatially into the cleaved reactive loop binding region in LSI-01 defined by the amino acids from about N185 to about T201, from about Q202 to about 1209, from about A217 to about K227, from about K368 to about V377, or some portion thereof, of SEQ ID NO:2 (corresponding to SEQ ID NO:27, 28, 29, or 30, respectively) in accordance with the structural coordinates of Table III.

Structure coordinates of the cleaved reactive loop binding region in LSI-01 defined by the amino acids from about N185 to about T201, from about Q202 to about 1209, from about A217 to about K227, and/or from about K368 to about V377 (SEQ ID NO:27, 28, 29, or 30, respectfully) and/or the heparin binding region in LSI-01 defined by the amino acids from about Y63 to about E80, from about E125 to about T140, and/or from about A306 to about S315 (SEQ ID NO:24, 25, or 26, respectfully), can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential LSI-01 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction, the three-dimensional structural model can be employed to design or select compounds as potential LSI-01 modulators. Compounds identified as potential LSI-01 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the LSI-01, or in characterizing the ability of LSI-01 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential LSI-01 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids N185-T201, Q202-I209, A217-K227, K368-V377, Y63-E80, E125-T140, A306-S315, A384,T385, A386, A387, T388, T389, T390, K391, F392, I393, V394, R395, S396, K397, D398, G399, P400, S401, Y402, F403, T404 from LSI-01 (SEQ ID NO:27, 28, 29, 30, 24, 25, 26, or 31, respectfully) in accordance with the structure coordinates of Table III.

However, as will be understood by those of skill in the art based upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.

For example, a number computer modeling systems are available in which the sequence of the LSI-01 and the LSI-01 structure (i.e., atomic coordinates of LSI-01 and/or the atomic coordinates of the reactive loop, the heparin binding region, and cleaved reactive loop binding region as provided in Table III) can be input. This computer system then generates the structural details of one or more these regions in which a potential LSI-01 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with LSI-01. In addition, the compound must be able to assume a conformation that allows it to associate with LSI-01. Some modeling systems estimate the potential inhibitory or binding effect of a potential LSI-01 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in one or more of the cleaved reactive loop binding region, the heparin binding region, or the reactive loop in LSI-01. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, their relationship to each other and LSI-01 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to CAVEAT (Bartlett et al. 1989) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, LSI-01 is overall well suited to modem methods including combinatorial chemistry.

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the heparin binding regions or the cleaved reactive loop region, and which may therefore be suitable candidates for synthesis and testing.

Additionally, the three-dimensional homology model of LSI-01 will aid in the design of mutants with altered biological activity. Such mutants may comprise conservative as well as non-conservative amino acid substitutions in any of the domains referenced herein.

The following are encompassed by the present invention: a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the model LSI-01 according to Table III or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of the complex of not more than 4.0 Å; and a machine-readable data storage medium, wherein said molecule is defined by the set of structure coordinates of the model for LSI-01 according to Table III, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 3.0 Å; a model comprising all or any part of the model defined by structure coordinates of LSI-01 according to Table III, or a mutant or homologue of said molecule or molecular complex.

In a further embodiment, the following are encompassed by the present invention: a method for identifying a mutant of LS1-01 with altered biological properties, function, or reactivity, the method comprising any combination of steps of: use of the model or a homologue of said model according to Table III, for the design of protein mutants with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; use of the model or a homologue of said model, for the design of a protein with mutations in the reactive loop region comprised of the amino acids A384,T385, A386, A387, T388, T389, T390, K391, F392, I393, V394, R395, S396, K397, D398, G399, P400, S401, Y402, F403, T404 according to Table III with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; use of the model or a homologue of said model, for the design of a protein with mutations in the heparin binding region comprised by the amino acids Y63-E80, E125-T140, A306-S315 according to Table III with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; and use of the model or a homologue of said model, for the design of a protein with mutations cleaved reaction loop binding region comprised by the amino acids N185-T201, Q202-1209, A217-K227, K368-V377 according to Table III , with altered biological function or properties which exhibit any combination of therapeutic effects outlined provided elsewhere herein.

In further preferred embodiments, the following are encompassed by the present invention: a method for identifying modulators of LSl-01 biological properties, function, or reactivity, the method comprising any combination of steps of: modeling test compounds that overlay spatially into the heparin bindings region defined by all or any portion of residues Y63-E80, E125-T140, A306-S315, of the three-dimensional structural model according to Table III, or using a homologue or portion thereof, modeling test compounds that overlay spatially into the regions defined by all or any portion of the cleaved reactive loop binding regions comprised of the residues N185-T201, Q202-1209, A217-K227, K368-V377 of the three-dimensional structural model according to Table III, or using a homologue or portion thereof;

The present invention encompasses using the structure coordinates as set forth herein to identify structural and chemical features of the LSI-01 polypeptide; employing identified structural or chemical features to design or select compounds as potential LSI-01 modulators; employing the three-dimensional structural model to design or select compounds as potential LSI-01 modulators; synthesizing the potential LSI-01 modulators; screening the potential LSI-01 modulators in an assay characterized by binding of a protein to the LSI-01; selecting the potential LSI-01 modulator from a database; designing the LSI-01 modulator de novo; and/or designing said LSI-01 modulator from a known modulator activity.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1752 of SEQ ID NO:1, b is an integer between 15 to 1766, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and where b is greater than or equal to a+14. TABLE I ATCC NT Total 5′ NT Deposit SEQ NT of Start 3′ NT AA Total Gene CDNA No. Z and ID. Seq of Codon of Seq ID AA of No. CloneID Date Vector No. X Clone of ORF ORF No. Y ORF 1. LSI-01 - PTA-2766 PSport1 1 1766 68 1372 2 435 (also Dec. 08, referred to 2000 as bac708.clone 24; AL132708_FL; BA- 14448; protease 6; and/or protease inhibitor 6)

Table I summarizes the information corresponding to each “Gene No.” described above. The nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the “cDNA clone ID” identified in Table I and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually several overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:1.

The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in “ATCC deposit No:PTA-2766 and Date.” “Vector” refers to the type of vector contained in the cDNA Clone ID.

“Total NT Seq. Of Clone” refers to the total number of nucleotides in the clone contig identified by “Gene No.” The deposited clone may contain all or most of the sequence of SEQ ID NO:1. The nucleotide position of SEQ ID NO:1 of the putative start codon (methionine) is identified as “5′ NT of Start Codon of ORF.”

The translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:2,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The total number of amino acids within the open reading frame of SEQ ID NO:2 is identified as “Total AA of ORF”.

SEQ ID NO:1 (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:2 (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further herein. For instance, SEQ ID NO:1 is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:1 or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:2 may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the proteins encoded by the cDNA clones identified in Table I.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides may cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:1 and the predicted translated amino acid sequence identified as SEQ ID NO:2, but also a sample of plasmid DNA containing a cDNA of the invention deposited with the ATCC, as set forth in Table I. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited cDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ ID NO:1, SEQ ID NO:2, or the deposited clone. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are species homologs, allelic variants, and/or orthologs. The skilled artisan could, using procedures well-known in the art, obtain the polynucleotide sequence corresponding to full-length genes (including, but not limited to the full-length coding region), allelic variants, splice variants, orthologs, and/or species homologues of genes corresponding to SEQ ID NO:1, SEQ ID NO:2, or a deposited clone, relying on the sequence from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologues may be isolated and identified by making suitable probes or primers which correspond to the 5′, 3′, or internal regions of the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the protein, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using protocols described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the full-length form of the protein.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, and/or a cDNA provided in ATCC Deposit No. Z:. The present invention also provides a polypeptide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:2, and/or a polypeptide encoded by the cDNA provided in ATCC deposit No:PTA-2766. The present invention also provides polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:2, and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit No:PTA-2766.

Preferably, the present invention is directed to a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, and/or a cDNA provided in ATCC Deposit No.: that is less than, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs in length.

The present invention encompasses polynucleotides with sequences complementary to those of the polynucleotides of the present invention disclosed herein. Such sequences may be complementary to the sequence disclosed as SEQ ID NO:1, the sequence contained in a deposit, and/or the nucleic acid sequence encoding the sequence disclosed as SEQ ID NO:2.

The present invention also encompasses polynucleotides capable of hybridizing, preferably under reduced stringency conditions, more preferably under stringent conditions, and most preferably under highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in Table II below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. TABLE II Strin- gency Hybridization Wash Con- Polynucleotide Hybrid Temperature Temperature dition Hybrid± Length (bp)‡ and Buffer† and Buffer† A DNA:DNA > or equal to 65° C.; 1xSSC - 65° C.; 50 or- 42° C.; 0.3xSSC 1xSSC, 50% formamide B DNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > or equal to 67° C.; 1xSSC - 67° C.; 50 or- 45° C.; 0.3xSSC 1xSSC, 50% formamide D DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 70° C.; 1xSSC - 70° C.; 50 or- 50° C.; 0.3xSSC 1xSSC, 50% formamide F RNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 65° C.; 4xSSC - 65° C.; 1xSSC 50 or- 45° C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA > or equal to 67° C.; 4xSSC - 67° C.; 1xSSC 50 or- 45° C.; 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA > or equal to 70° C.; 4xSSC - 67° C.; 1xSSC 50 or- 40° C.; 6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA > or equal to 50° C.; 4xSSC - 50° C.; 2xSSC 50 or- 40° C. 6xSSC, 50% formamide N DNA:DNA <50 Tn*; 6xSSC Tn*; 6xSSC O DNA:RNA > or equal to 55° C.; 4xSSC - 55° C.; 2xSSC 50 or- 42° C.; 6xSSC, 50% formamide P DNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 60° C.; 4xSSC - 60° C.; 2xSSC 50 or- 45° C.; 6xSSC, 50% formamide R RNA:RNA <50 Tr*; 4xSSC Tr*; 4xSSC ‡The “hybrid length” is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucleotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence # complementarity. Methods of aligning two or more polynucleotide sequences and/or determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA * Star suite of programs, etc). †SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. The hydridizations and washes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide. *Tb-Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less # than 18 base pairs in length, Tm(° C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.) = # 81.5 + 16.6(log₁₀[Na+]) + 0.41 (% G + C) − (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1xSSC = .165M). ±The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified polynucleotide. Such modified polynucleotides are known in the art and are more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotide hybridization are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby incorporated by reference herein.

Preferably, such hybridizing polynucleotides have at least 70% sequence identity (more preferably, at least 80% identity; and most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which they hybridize, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The determination of identity is well known in the art, and discussed more specifically elsewhere herein.

The invention encompasses the application of PCR methodology to the polynucleotide sequences of the present invention, the clone deposited with the ATCC, and/or the cDNA encoding the polypeptides of the present invention. PCR techniques for the amplification of nucleic acids are described in U.S. Pat. No. 4, 683, 195 and Saiki et al., Science, 239:487-491 (1988). PCR, for example, may include the following steps, of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerization. The nucleic acid probed or used as a template in the amplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA. References for the general use of PCR techniques, including specific method parameters, include Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds., Innis et al., Academic Press, New York, (1990).

Signal Sequences

The present invention also encompasses mature forms of the polypeptide comprising, or alternatively consisting of, the polypeptide sequence of SEQ ID NO:2, the polypeptide encoded by the polynucleotide described as SEQ ID NO:1, and/or the polypeptide sequence encoded by a cDNA in the deposited clone. The present invention also encompasses polynucleotides encoding mature forms of the present invention, such as, for example the polynucleotide sequence of SEQ ID NO:1, and/or the polynucleotide sequence provided in a cDNA of the deposited clone.

According to the signal hypothesis, proteins secreted by eukaryotic cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most eukaryotic cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

The established method for identifying the location of signal sequences, in addition, to their cleavage sites has been the SignalP program (v1.1) developed by Henrik Nielsen et al., Protein Engineering 10:1-6 (1997). The program relies upon the algorithm developed by von Heinje, though provides additional parameters to increase the prediction accuracy.

More recently, a hidden Markov model has been developed (H. Neilson, et al., Ismb 1998;6:122-30), which has been incorporated into the more recent SignalP (v2.0). This new method increases the ability to identify the cleavage site by discriminating between signal peptides and uncleaved signal anchors. The present invention encompasses the application of the method disclosed therein to the prediction of the signal peptide location, including the cleavage site, to any of the polypeptide sequences of the present invention.

As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the polypeptide of the present invention may contain a signal sequence. Polypeptides of the invention which comprise a signal sequence have an N-terminus beginning within 5 residues (i.e., +or −5 residues, or preferably at the −5, −4, −3, −2, −1, +1, +2, +3, +4, or +5 residue) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:1 and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants, orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQ ID NO:1, the complementary strand thereto, and/or the cDNA sequence contained in the deposited clone.

The present invention also encompasses variants of the polypeptide sequence, and/or fragments therein, disclosed in SEQ ID NO:2, a polypeptide encoded by the polynucleotide sequence in SEQ ID NO:1, and/or a polypeptide encoded by a cDNA in the deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a LSI-01 related polypeptide having an amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:PTA-2766; (b) a nucleotide sequence encoding a mature LSI-01 related polypeptide having the amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:PTA-2766; (c) a nucleotide sequence encoding a biologically active fragment of a LSI-01 related polypeptide having an amino acid sequence shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:PTA-2766; (d) a nucleotide sequence encoding an antigenic fragment of a LSI-01 related polypeptide having an amino acid sequence sown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:PTA-2766; (e) a nucleotide sequence encoding a LSI-01 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:PTA-2766; (f) a nucleotide sequence encoding a mature LSI-01 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:PTA-2766; (g) a nucleotide sequence encoding a biologically active fragment of a LSI-01 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:PTA-2766; (h) a nucleotide sequence encoding an antigenic fragment of a LSI-01 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:PTA-2766; (I) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention is also directed to polynucleotide sequences which comprise, or alternatively consist of, a polynucleotide sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.

Another aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively, consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a LSI-01 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (b) a nucleotide sequence encoding a mature LSI-01 related polypeptide having the amino acid sequence as shown in the sequence listing and descried in Table I; (c) a nucleotide sequence encoding a biologically active fragment of a LSI-01 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (d) a nucleotide sequence encoding an antigenic fragment of a LSI-01 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (e) a nucleotide sequence encoding a LSI-01 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (f) a nucleotide sequence encoding a mature LSI-01 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I: (g) a nucleotide sequence encoding a biologically active fragment of a LSI-01 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (h) a nucleotide sequence encoding an antigenic fragment of a LSI-01 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC deposit and described in Table I; (i) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.

The present invention is also directed to nucleic acid molecules which comprise, or alternatively, consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention encompasses polypeptide sequences which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 98%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the following non-limited examples, the polypeptide sequence identified as SEQ ID NO:2, the polypeptide sequence encoded by a cDNA provided in the deposited clone, and/or polypeptide fragments of any of the polypeptides provided herein. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.

The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 98%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO:2, a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:1, a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these polypeptides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompasses by the present invention, as are the polypeptides encoded by these polynucleotides.

By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referenced in Table I, the ORF (open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. However, the CLUSTALW algorithm automatically converts U's to T's when comparing RNA sequences to DNA sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polynucleotide alignment. Percent identity calculations based upon global polynucleotide alignments are often preferred since they reflect the percent identity between the polynucleotide molecules as a whole (i.e., including any polynucleotide overhangs, not just overlapping regions), as opposed to, only local matching polynucleotides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the CLUSTALW alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.

In addition to the above method of aligning two or more polynucleotide or polypeptide sequences to arrive at a percent identity value for the aligned sequences, it may be desirable in some circumstances to use a modified version of the CLUSTALW algorithm which takes into account known structural features of the sequences to be aligned, such as for example, the SWISS-PROT designations for each sequence. The result of such a modifed CLUSTALW algorithm may provide a more accurate value of the percent identity for two polynucleotide or polypeptide sequences. Support for such a modified version of CLUSTALW is provided within the CLUSTALW algorithm and would be readily appreciated to one of skill in the art of bioinformatics.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem... 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem.. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the protein will likely be retained when less than the majority of the residues of the protein are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptide of the present invention may, in fact, result in a significant increase in one or more of the biological activities of the polypeptide(s). For example, biological activity of many polypeptides are governed by the presence of regulatory domains at either one or both termini. Such regulatory domains effectively inhibit the biological activity of such polypeptides in lieu of an activation event (e.g., binding to a cognate ligand or receptor, phosphorylation, proteolytic processing, etc.). Thus, by eliminating the regulatory domain of a polypeptide, the polypeptide may effectively be rendered biologically active in the absence of an activation event.

Thus, the invention further includes polypeptide variants that show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.

The invention encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptide of the present invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics (e.g., chemical properties). According to Cunningham et al above, such conservative substitutions are likely to be phenotypically silent. Additional guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Tolerated conservative amino acid substitutions of the present invention involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservative substitutions provided in Table IV below. TABLE IV For Amino Acid Code Replace with any of: Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions may also increase protein or peptide stability. The invention encompasses amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.

Both identity and similarity can be readily calculated by reference to the following publications: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Informatics Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press,New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. Such probabilities are determined by aligning multiple genes with related function and assessing the relative penalty of each substitution to proper gene function. Such probabilities are often described in a matrix and are used by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percent similarity wherein similarity refers to the degree by which one amino acid may substitute for another amino acid without lose of function. An example of such a matrix is the PAM250 or BLOSUM62 matrix.

Aside from the canonical chemically conservative substitutions referenced above, the invention also encompasses substitutions which are typically not classified as conservative, but that may be chemically conservative under certain circumstances. Analysis of enzymatic catalysis for proteases, for example, has shown that certain amino acids within the active site of some enzymes may have highly perturbed pKa's due to the unique microenvironment of the active site. Such perturbed pKa's could enable some amino acids to substitute for other amino acids while conserving enzymatic structure and function. Examples of amino acids that are known to have amino acids with perturbed pKa's are the Glu-35 residue of Lysozyme, the Ue-16 residue of Chymotrypsin, the His-159 residue of Papain, etc. The conservation of function relates to either anomalous protonation or anomalous deprotonation of such amino acids, relative to their canonical, non-perturbed pKa. The pKa perturbation may enable these amino acids to actively participate in general acid-base catalysis due to the unique ionization environment within the enzyme active site. Thus, substituting an amino acid capable of serving as either a general acid or general base within the microenvironment of an enzyme active site or cavity, as may be the case, in the same or similar capacity as the wild-type amino acid, would effectively serve as a conservative amino substitution.

Besides conservative amino acid substitution, variants of the present invention include, but are not limited to, the following: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Moreover, the invention further includes polypeptide variants created through the application of molecular evolution (“DNA Shuffling”) methodology to the polynucleotide disclosed as SEQ ID NO:1, the sequence of the clone submitted in a deposit, and/or the cDNA encoding the polypeptide disclosed as SEQ ID NO:2. Such DNA Shuffling technology is known in the art and more particularly described elsewhere herein (e.g., WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples provided herein).

A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.

Polynucleotide and Polvpeptide Fragments

The present invention is directed to polynucleotide fragments of the polynucleotides of the invention, in addition to polypeptides encoded therein by said polynucleotides and/or fragments.

In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:1 or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:1. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about -nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:1, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Also encompassed by the present invention are polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, as are the polypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:2 or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments include the full-length protein. Further preferred polypeptide fragments include the full-length protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of the full-length polypeptide. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the full-length protein. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:2 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

In a preferred embodiment, the functional activity displayed by a polypeptide encoded by a polynucleotide fragment of the invention may be one or more biological activities typically associated with the full-length polypeptide of the invention. Illustrative of these biological activities includes the fragments ability to bind to at least one of the same antibodies which bind to the full-length protein, the fragments ability to interact with at lease one of the same proteins which bind to the full-length, the fragments ability to elicit at least one of the same immune responses as the full-length protein (i.e., to cause the immune system to create antibodies specific to the same epitope, etc.), the fragments ability to bind to at least one of the same polynucleotides as the full-length protein, the fragments ability to bind to a receptor of the full-length protein, the fragments ability to bind to a ligand of the full-length protein, and the fragments ability to multimerize with the full-length protein. However, the skilled artisan would appreciate that some fragments may have biological activities which are desirable and directly inapposite to the biological activity of the full-length protein. The functional activity of polypeptides of the invention, including fragments, variants, derivatives, and analogs thereof can be determined by numerous methods available to the skilled artisan, some of which are described elsewhere herein.

The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:1 or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:1), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal or plant, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med.. 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous arnino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homologue of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologues of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6M, 5×10−7 M, 107 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem... 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitable method known in the art.

The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed. (1988), which is hereby incorporated herein by reference in its entirety). For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. The administration of the polypeptides of the present invention may entail one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. For the purposes of the invention, “immunizing agent” may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV). The immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivitizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

The antibodies of the present invention may comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed. (1988), by Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., (1981)), or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies includes, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include polypeptides of the present invention or a fusion protein thereof. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thyrirdine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptides of the present invention. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term “monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. The DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). The hydridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples herein. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. The techniques of cole et al., and Boerder et al., are also available for the preparation of human monoclonal antibodies (cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and creation of an antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in the following scientific publications: Marks et al., Biotechnol., 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al., Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93 (1995).

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards a polypeptide of the present invention, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/20373; and EP03089). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem . . . 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:2 may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem . . . 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretharnine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

The present invention also encompasses the creation of synthetic antibodies directed against the polypeptides of the present invention. One example of synthetic antibodies is described in Radrizzani, M., et al., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class of synthetic antibodies has been described and are referred to as molecularly imprinted polymers (MEPs) (Semorex, Inc.). Antibodies, peptides, and enzymes are often used as molecular recognition elements in chemical and biological sensors. However, their lack of stability and signal transduction mechanisms limits their use as sensing devices. Molecularly imprinted polymers (MIPs) are capable of mimicking the function of biological receptors but with less stability constraints. Such polymers provide high sensitivity and selectivity while maintaining excellent thermal and mechanical stability. MIPs have the ability to bind to small molecules and to target molecules such as organics and proteins' with equal or greater potency than that of natural antibodies. These “super” MIPs have higher affinities for their target and thus require lower concentrations for efficacious binding.

During synthesis, the MIPs are imprinted so as to have complementary size, shape, charge and functional groups of the selected target by using the target molecule itself (such as a polypeptide, antibody, etc.), or a substance having a very similar structure, as its “print” or “template.” MIPs can be derivatized with the same reagents afforded to antibodies. For example, fluorescent ‘super’ MIPs can be coated onto beads or wells for use in highly sensitive separations or assays, or for use in high throughput screening of proteins.

Moreover, MIPs based upon the structure of the polypeptide(s) of the present invention may be useful in screening for compounds that bind to the polypeptide(s) of the invention. Such a MIP would serve the role of a synthetic “receptor” by minimicking the native architecture of the polypeptide. In fact, the ability of a MIP to serve the role of a synthetic receptor has already been demonstrated for the estrogen receptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001); Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71, (2001)). A synthetic receptor may either be mimicked in its entirety (e.g., as the entire protein), or mimicked as a series of short peptides corresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys, Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may be employed in any one or more of the screening methods described elsewhere herein.

MIPs have also been shown to be useful in “sensing” the presence of its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron., 16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001)). For example, a MIP designed using a polypeptide of the present invention may be used in assays designed to identify, and potentially quantitate, the level of said polypeptide in a sample. Such a MIP may be used as a substitute for any component described in the assays, or kits, provided herein (e.g., ELISA, etc.).

A number of methods may be employed to create MIPs to a specific receptor, ligand, polypeptide, peptide, organic molecule. Several preferred methods are described by Esteban et al in J. Anal, Chem., 370(7):795-802, (2001), which is hereby incorporated herein by reference in its entirety in addition to any references cited therein. Additional methods are known in the art and are encompassed by the present invention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem, Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J., De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54, (2001); which are hereby incorporated by reference in their entirety herein.

Uses for Antibodies Directed Against Polypeptides of the Invention

The antibodies of the present invention have various utilities. For example, such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample. Such a diagnostic assay may be comprised of at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or a chromatography column, etc. And a second step involving the quantification of antibody bound to the substrate. Alternatively, the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., (1987), ppl47-158). The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219 (1981); and Nygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present invention are useful for the affinity purification of such polypeptides from recombinant cell culture or natural sources. In this process, the antibodies against a particular polypeptide are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the polypeptides to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except for the desired polypeptides, which are bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the desired polypeptide from the antibody.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sephardse beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6 M, 5×10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Antibodies directed against polypeptides of the present invention are useful for inhibiting allergic reactions in animals. For example, by administering a therapeutically acceptable dose of an antibody, or antibodies, of the present invention, or a cocktail of the present antibodies, or in combination with other antibodies of varying sources, the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the gene encoding an antibody directed against a polypeptide of the present invention, said polypeptide having the potential to elicit an allergic and/or immune response in an organism, and transforming the organism with said antibody gene such that it is expressed (e.g., constitutively, inducibly, etc.) in the organism. Thus, the organism would effectively become resistant to an allergic response resulting from the ingestion or presence of such an immune/allergic reactive polypeptide. Moreover, such a use of the antibodies of the present invention may have particular utility in preventing and/or ameliorating autoimmune diseases and/or disorders, as such conditions are typically a result of antibodies being directed against endogenous proteins. For example, in the instance where the polypeptide of the present invention is responsible for modulating the immune response to auto-antigens, transforming the organism and/or individual with a construct comprising any of the promoters disclosed herein or otherwise known in the art, in addition, to a polynucleotide encoding the antibody directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the auto-antigen(s). Detailed descriptions of therapeutic and/or gene therapy applications of the present invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced in a plant (e.g., cloning the gene of the antibody directed against a polypeptide of the present invention, and transforming a plant with a suitable vector comprising said gene for constitutive expression of the antibody within the plant), and the plant subsequently ingested by an animal, thereby conferring temporary immunity to the animal for the specific antigen the antibody is directed towards (See, for example, U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferably polyclonal antibodies, more preferably monoclonal antibodies, and most preferably single-chain antibodies, can be used as a means of inhibiting gene expression of a particular gene, or genes, in a human, mammal, and/or other organism. See, for example, International Publication Number WO 00/05391, published Feb. 2, 2000, to Dow Agrosciences LLC. The application of such methods for the antibodies of the present invention are known in the art, and are more particularly described elsewhere herein.

In yet another embodiment, antibodies of the present invention may be useful for multimerizing the polypeptides of the present invention. For example, certain proteins may confer enhanced biological activity when present in a multimeric state (i.e., such enhanced activity may be due to the increased effective concentration of such proteins whereby more protein is available in a localized location).

Antibody-Based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem . . . 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; W092/20316; W093/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication W094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging With Antibodies

Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Kits

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because certain proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Similarly, peptide cleavage sites can be introduced in-between such peptide moieties, which could additionally be subjected to protease activity to remove said peptide(s) from the protein of the present invention. The addition of peptide moieties, including peptide cleavage sites, to facilitate handling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem . . . 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused to marker sequences (also referred to as “tags” ). Due to the availability of antibodies specific to such “tags”, purification of the fused polypeptide of the invention, and/or its identification is significantly facilitated since antibodies specific to the polypeptides of the invention are not required. Such purification may be in the form of an affinity purification whereby an anti-tag antibody or another type of affinity matrix (e.g., anti-tag antibody attached to the matrix of a flow-thru column) that binds to the epitope tag is present. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984)).

The skilled artisan would acknowledge the existence of other “tags” which could be readily substituted for the tags referred to supra for purification and/or identification of polypeptides of the present invention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). For example, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology 5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553 (1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ ID NO:33), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitope peptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitope peptide (Skinner et al., J. Biol. Chem . . . , 266:15136-15166, (1991)); the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFP epitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

The present invention also encompasses the attachment of up to nine codons encoding a repeating series of up to nine arginine amino acids to the coding region of a polynucleotide of the present invention. The invention also encompasses chemically derivitizing a polypeptide of the present invention with a repeating series of up to nine arginine amino acids. Such a tag, when attached to a polypeptide, has recently been shown to serve as a universal pass, allowing compounds access to the interior of cells without additional derivitization or manipulation (Wender, P., et al., unpublished data).

Protein fusions involving polypeptides of the present invention, including fragments and/or variants thereof, can be used for the following, non-limiting examples, subcellular localization of proteins, determination of protein-protein interactions via immunoprecipitation, purification of proteins via affinity chromatography, functional and/or structural characterization of protein. The present invention also encompasses the application of hapten specific antibodies for any of the uses referenced above for epitope fusion proteins. For example, the polypeptides of the present invention could be chemically derivatized to attach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to the availability of monoclonal antibodies specific to such haptens, the protein could be readily purified using immunoprecipation, for example.

Polypeptides of the present invention, including fragments and/or variants thereof, in addition to, antibodies directed against such polypeptides, fragments, and/or variants, may be fused to any of a number of known, and yet to be determined, toxins, such as ricin, saporin (Mashiba H, et al., Ann. N.Y. Acad. Sci. 1999; 886:233-5), or HC toxin (Tonukari N J, et al., Plant Cell. February 2000; 12(2):237-248), for example. Such fusions could be used to deliver the toxins to desired tissues for which a ligand or a protein capable of binding to the polypeptides of the invention exists.

The invention encompasses the fusion of antibodies directed against polypeptides of the present invention, including variants and fragments thereof, to said toxins for delivering the toxin to specific locations in a cell, to specific tissues, and/or to specific species. Such bifunctional antibodies are known in the art, though a review describing additional advantageous fusions, including citations for methods of production, can be found in P. J. Hudson, Curr. Opp. In. Imm. 11:548-557, (1999); this publication, in addition to the references cited therein, are hereby incorporated by reference in their entirety herein. In this context, the term “toxin” may be expanded to include any heterologous protein, a small molecule, radionucleotides, cytotoxic drugs, liposomes, adhesion molecules, glycoproteins, ligands, cell or tissue-specific ligands, enzymes, of bioactive agents, biological response modifiers, anti-fungal agents, hormones, steroids, vitamins, peptides, peptide analogs, anti-allergenic agents, anti-tubercular agents, anti-viral agents, antibiotics, anti-protozoan agents, chelates, radioactive particles, radioactive ions, X-ray contrast agents, monoclonal antibodies, polyclonal antibodies and genetic material. In view of the present disclosure, one skilled in the art could determine whether any particular “toxin” could be used in the compounds of the present invention. Examples of suitable “toxins” listed above are exemplary only and are not intended to limit the “toxins” that may be used in the present invention.

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,”D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDI, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG, as required.

In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein, the addition of epitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin, maltose binding protein, etc.), attachment of affinity tags such as biotin and/or streptavidin, the covalent attachment of chemical moieties to the amino acid backbone, N- or C-terminal processing of the polypeptides ends (e.g., proteolytic processing), deletion of the N-terminal methionine residue, etc.

Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The invention further encompasses chemical derivitization of the polypeptides of the present invention, preferably where the chemical is a hydrophilic polymer residue. Exemplary hydrophilic polymers, including derivatives, may be those that include polymers in which the repeating units contain one or more hydroxy groups (polyhydroxy polymers), including, for example, poly(vinyl alcohol); polymers in which the repeating units contain one or more amino groups (polyamine polymers), including, for example, peptides, polypeptides, proteins and lipoproteins, such as albumin and natural lipoproteins; polymers in which the repeating units contain one or more carboxy groups (polycarboxy polymers), including, for example, carboxymethylcellulose, alginic acid and salts thereof, such as sodium and calcium alginate, glycosaminoglycans and salts thereof, including salts of hyaluronic acid, phosphorylated and sulfonated derivatives of carbohydrates, genetic material, such as interleukin-2 and interferon, and phosphorothioate oligomers; and polymers in which the repeating units contain one or more saccharide moieties (polysaccharide polymers), including, for example, carbohydrates.

The molecular weight of the hydrophilic polymers may vary, and is generally about 50 to about 5,000,000, with polymers having a molecular weight of about 100 to about 50,000 being preferred. The polymers may be branched or unbranched. More preferred polymers have a molecular weight of about 150 to about 10,000, with molecular weights of 200 to about 8,000 being even more preferred.

For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

Additional preferred polymers which may be used to derivatize polypeptides of the invention, include, for example, poly(ethylene glycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate and poly(vinyl alcohol), with PEG polymers being particularly preferred. Preferred among the PEG polymers are PEG polymers having a molecular weight of from about 100 to about 10,000. More preferably, the PEG polymers have a molecular weight of from about 200 to about 8,000, with PEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of 2,000, 5,000 and 8,000, respectively, being even more preferred. Other suitable hydrophilic polymers, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, the polymers used may include polymers that can be attached to the polypeptides of the invention via alkylation or acylation reactions.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As with the various polymers exemplified above, it is contemplated that the polymeric residues may contain functional groups in addition, for example, to those typically involved in linking the polymeric residues to the polypeptides of the present invention. Such functionalities include, for example, carboxyl, amine, hydroxy and thiol groups. These functional groups on the polymeric residues can be further reacted, if desired, with materials that are generally reactive with such functional groups and which can assist in targeting specific tissues in the body including, for example, diseased tissue. Exemplary materials which can be reacted with the additional functional groups include, for example, proteins, including antibodies, carbohydrates, peptides, glycopeptides, glycolipids, lectins, and nucleosides.

In addition to residues of hydrophilic polymers, the chemical used to derivatize the polypeptides of the present invention can be a saccharide residue. Exemplary saccharides which can be derived include, for example, monosaccharides or sugar alcohols, such as erythrose, threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol and sedoheptulose, with preferred monosaccharides being fructose, mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides, such as lactose, sucrose, maltose and cellobiose. Other saccharides include, for example, inositol and ganglioside head groups. Other suitable saccharides, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, saccharides which may be used for derivitization include saccharides that can be attached to the polypeptides of the invention via alkylation or acylation reactions.

Moreover, the invention also encompasses derivitization of the polypeptides of the present invention, for example, with lipids (including cationic, anionic, polymerized, charged, synthetic, saturated, unsaturated, and any combination of the above, etc.). stabilizing agents.

The invention encompasses derivitization of the polypeptides of the present invention, for example, with compounds that may serve a stabilizing function (e.g., to increase the polypeptides half-life in solution, to make the polypeptides more water soluble, to increase the polypeptides hydrophilic or hydrophobic character, etc.). Polymers useful as stabilizing materials may be of natural, semi-synthetic (modified natural) or synthetic origin. Exemplary natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof Accordingly, suitable polymers include, for example, proteins, such as albumin, polyalginates, and polylactide-coglycolide polymers. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol (including for example, the class of compounds referred to as Pluronics.RTM., commercially available from BASF, Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof. Methods for the preparation of derivatized polypeptides of the invention which employ polymers as stabilizing compounds will be readily apparent to one skilled in the art, in view of the present disclosure, when coupled with information known in the art, such as that described and referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporated by reference herein in its entirety.

Moreover, the invention encompasses additional modifications of the polypeptides of the present invention. Such additional modifications are known in the art, and are specifically provided, in addition to methods of derivitization, etc., in U.S. Pat. No. 6,028,066, which is hereby incorporated in its entirety herein.

The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:2 or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.

In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.

Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.

In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

In addition, the polynucleotide insert of the present invention could be operatively linked to “artificial” or chimeric promoters and transcription factors. Specifically, the artificial promoter could comprise, or alternatively consist, of any combination of cis-acting DNA sequence elements that are recognized by trans-acting transcription factors. Preferably, the cis acting DNA sequence elements and trans-acting transcription factors are operable in mammals. Further, the trans-acting transcription factors of such “artificial” promoters could also be “artificial” or chimeric in design themselves and could act as activators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:1. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:1 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Disease mapping data are known in the art. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.

Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected organisms can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected organisms, but not in normal organisms, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal organisms is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected organisms as compared to unaffected organisms can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an organism and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.

By “measuring the expression level of a polynucleotide of the present invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of organisms not having a disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an organism, body fluids, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as the following non-limiting examples, sputum, amniotic fluid, urine, saliva, breast milk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from organisms are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

The method(s) provided above may Preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including proliferative diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supra are hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the stronger binding characteristics of PNA:DNA hybrids. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.

In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat or prevent disease.

The present invention encompasses the addition of a nuclear localization signal, operably linked to the 5′ end, 3′ end, or any location therein, to any of the oligonucleotides, antisense oligonucleotides, triple helix oligonucleotides, ribozymes, PNA oligonucleotides, and/or polynucleotides, of the present invention. See, for example, G. Cutrona, et al., Nat. Biotech., 18:300-303, (2000); which is hereby incorporated herein by reference.

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. In one example, polynucleotide sequences of the present invention may be used to construct chimeric RNA/DNA oligonucleotides corresponding to said sequences, specifically designed to induce host cell mismatch repair mechanisms in an organism upon systemic injection, for example (Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which is hereby incorporated by reference herein in its entirety). Such RNA/DNA oligonucleotides could be designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes in the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc.). Alternatively, the polynucleotide sequence of the present invention may be used to construct duplex oligonucleotides corresponding to said sequence, specifically designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes into the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc). Such methods of using duplex oligonucleotides are known in the art and are encompassed by the present invention (see EP1007712, which is hereby incorporated by reference herein in its entirety).

The polynucleotides are also useful for identifying organisms from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an organisms genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, organisms can be identified because each organism will have a unique set of DNA sequences. Once an unique ID database is established for an organism, positive identification of that organism, living or dead, can be made from extremely small tissue samples. Similarly, polynucleotides of the present invention can be used as polymorphic markers, in addition to, the identification of transformed or non-transformed cells and/or tissues.

There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Moreover, as mentioned above, such reagents can be used to screen and/or identify transformed and non-transformed cells and/or tissues.

In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Moreover, polypeptides of the present invention can be used to treat, prevent, and/or diagnose disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor suppressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3:31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.

As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

The polynucleotide vector constructs of the invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.

In certain embodiments, the polynucleotide constructs of the invention are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA , 86:6077-6081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem . . . 265:10189-10192 (1990), which is herein incorporated by reference), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology, 101:512-527 (1983), which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell , 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA , 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem . . . , 255:10431 (1980); Szoka et al., Proc. Natl. Acad. Sci. USA , 75:145 (1978); Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides of the invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy , 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell , 68:143-155 (1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct containing polynucleotides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.

Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.

The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.

The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.

The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding angiogenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.

Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers. (Kaneda et al., Science, 243:375 (1989)).

A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA, 189:11277-11281 (1992), which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian. Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer or some autoimmune diseases, disorders, and/or conditions, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. A polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein diseases, disorders, and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, arterial thrombosis, venous thrombosis, etc.), blood platelet diseases, disorders, and/or conditions (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. Polynucleotides or polypeptides, or agonists or antagonists of the present invention are may also be useful for the detection, prognosis, treatment, and/or prevention of heart attacks (infarction), strokes, scarring, fibrinolysis, uncontrolled bleeding, uncontrolled coagulation, uncontrolled complement fixation, and/or inflammation.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be useful in treating, preventing, and/or diagnosing autoimmune diseases, disorders, and/or conditions. Many autoimmune diseases, disorders, and/or conditions result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune diseases, disorders, and/or conditions.

Examples of autoimmune diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to treat, prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.

Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate inflammation. For example, the polypeptide or polynucleotide or agonists or antagonist may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat, prevent, and/or diagnose inflammatory conditions, both chronic and acute conditions, including chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat, prevent, and/or diagnose hyperproliferative diseases, disorders, and/or conditions, including neoplasms. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative diseases, disorders, and/or conditions can be treated, prevented, and/or diagnosed. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating, preventing, and/or diagnosing hyperproliferative diseases, disorders, and/or conditions, such as a chemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative diseases, disorders, and/or conditions can also be treated, prevented, and/or diagnosed by a polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative diseases, disorders, and/or conditions include, but are not limited to: hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/or conditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

One preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating or preventing cell proliferative diseases, disorders, and/or conditions by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method of treating or preventing cell-proliferative diseases, disorders, and/or conditions in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the polynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96:324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes ” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.

For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.

Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating, preventing, and/or diagnosing one or more of the described diseases, disorders, and/or conditions. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the present invention are useful for treating, preventing, and/or diagnosing a subject having or developing cell proliferative and/or differentiation diseases, disorders, and/or conditions as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of diseases, disorders, and/or conditions related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-6M, 10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-10M, 10-10M, 5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M, 5×10-15M, and 10-15M.

Moreover, polypeptides of the present invention may be useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).

Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins, antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem. Biol. Interact. April 24; 111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int. J. Tissue React. 20(1):3-15 (1998), which are all hereby incorporated by reference).

Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewhere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such therapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of the invention may be used to treat, prevent, and/or diagnose cardiovascular diseases, disorders, and/or conditions, including peripheral artery disease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders, and/or conditions, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of the invention, are especially effective for the treatment of critical limb ischemia and coronary disease.

Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides of the invention are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye diseases, disorders, and/or conditions, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).

The present invention provides for treatment of diseases, disorders, and/or conditions associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).Thus, the present invention provides a method of treating, preventing, and/or diagnosing an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treat or prevent a cancer or tumor. Cancers which may be treated, prevented, and/or diagnosed with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat or prevent cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating, preventing, and/or diagnosing other diseases, disorders, and/or conditions, besides cancers, which involve angiogenesis. These diseases, disorders, and/or conditions include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods are provided for treating, preventing, and/or diagnosing hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating, preventing, and/or diagnosing neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.

Moreover, Ocular diseases, disorders, and/or conditions associated with neovascularization which can be treated, prevented, and/or diagnosed with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).

Thus, within one aspect of the present invention methods are provided for treating or preventing neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of diseases, disorders, and/or conditions can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided for treating or preventing neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat or prevent early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating or preventing proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided for treating or preventing retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.

Additionally, diseases, disorders, and/or conditions which can be treated, prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, diseases, disorders, and/or conditions and/or states, which can be treated, prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. In preferred embodiments, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cell survival that could be treated, prevented or diagnosed by the polynucleotides or polypeptides, or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative diseases, disorders, and/or conditions (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestablishment subsequent to dermal loss The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are a non-exhaustive list of grafts that polynucleotides or polypeptides, agonists or antagonists of the invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepidermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to promote skin strength and to improve the appearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat diseases associate with the under expression of the polynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. A growth factor such as the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated, prevented, and/or diagnosed using the polynucleotides or polypeptides, and/or agonists or antagonists of the invention. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).

In addition, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can be treated, prevented, and/or diagnosed with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases, disorders, and/or conditions which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated, prevented, and/or diagnosed in a patient (including human and non-human mammalian patients) according to the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases, disorders, and/or conditions, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral hypoxia. In one aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral ischemia. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral infarction. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose or prevent neural cell injury associated with a stroke. In a further aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981)); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/or conditions that may be treated, prevented, and/or diagnosed according to the invention include, but are not limited to, diseases, disorders, and/or conditions such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as diseases, disorders, and/or conditions that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat, prevent, and/or diagnose infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated, prevented, and/or diagnosed. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide or polynucleotide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated, prevented, and/or diagnosed include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated, prevented, and/or diagnosed using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic diseases, disorders, and/or conditions (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated, prevented, and/or diagnosed using the polynucleotide or polypeptide and/or agonist or antagonist of the present invention.

Chemotaxis

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat, prevent, and/or diagnose inflammation, infection, hyperproliferative diseases, disorders, and/or conditions, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat, prevent, and/or diagnose wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat, prevent, and/or diagnose wounds.

It is also contemplated that a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may inhibit chemotactic activity. These molecules could also be used to treat, prevent, and/or diagnose diseases, disorders, and/or conditions. Thus, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors),or small molecules.

Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.

Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of polypeptides of the invention thereby effectively generating agonists and antagonists of polypeptides of the invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides of the invention may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired polynucleotide sequence of the invention molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptides of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of the polypeptides of the invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat, prevent, and/or diagnose disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues. Therefore, the invention includes a method of identifying compounds which bind to the polypeptides of the invention comprising the steps of: (a) incubating a candidate binding compound with the polypeptide; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with the polypeptide, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the invention experimentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence. Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, any combination or all of contained in the polypeptide sequences of the invention. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention.

Targeted Delivery

In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.

As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.

This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.

The human LSI-1 polypeptides and/or peptides of the present invention, or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic drugs or compounds in a variety of drug screening techniques. The fragment employed in such a screening assay may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The reduction or abolition of activity of the formation of binding complexes between the ion channel protein and the agent being tested can be measured. Thus, the present invention provides a method for screening or assessing a plurality of compounds for their specific binding affinity with a LSI-1 polypeptide, or a bindable peptide fragment, of this invention, comprising providing a plurality of compounds, combining the LSI-1 polypeptide, or a bindable peptide fragment, with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions and detecting binding of the LSI-1 polypeptide or peptide to each of the plurality of test compounds, thereby identifying the compounds that specifically bind to the LSI-1 polypeptide or peptide.

Methods of identifying compounds that modulate the activity of the novel human LSI-1 polypeptides and/or peptides are provided by the present invention and comprise combining a potential or candidate compound or drug modulator of calpain biological activity with an LSI-1 polypeptide or peptide, for example, the LSI-1 amino acid sequence as set forth in SEQ ID NOS:2, and measuring an effect of the candidate compound or drug modulator on the biological activity of the LSI-1 polypeptide or peptide. Such measurable effects include, for example, physical binding interaction; the ability to cleave a suitable calpain substrate; effects on native and cloned LSI-1-expressing cell line; and effects of modulators or other calpain-mediated physiological measures.

Another method of identifying compounds that modulate the biological activity of the novel LSI-1 polypeptides of the present invention comprises combining a potential or candidate compound or drug modulator of a calpain biological activity with a host cell that expresses the LSI-1 polypeptide and measuring an effect of the candidate compound or drug modulator on the biological activity of the LSI-1 polypeptide. The host cell can also be capable of being induced to express the LSI-1 polypeptide, e.g., via inducible expression. Physiological effects of a given modulator candidate on the LSI-1 polypeptide can also be measured. Thus, cellular assays for particular calpain modulators may be either direct measurement or quantification of the physical biological activity of the LSI-1 polypeptide, or they may be measurement or quantification of a physiological effect. Such methods preferably employ a LSI-1 polypeptide as described herein, or an overexpressed recombinant LSI-1 polypeptide in suitable host cells containing an expression vector as described herein, wherein the LSI-1 polypeptide is expressed, overexpressed, or undergoes upregulated expression.

Another aspect of the present invention embraces a method of screening for a compound that is capable of modulating the biological activity of a LSI-1 polypeptide, comprising providing a host cell containing an expression vector harboring a nucleic acid sequence encoding a LSI-1 polypeptide, or a functional peptide or portion thereof (e.g., SEQ ID NOS:2); determining the biological activity of the expressed LSI-1 polypeptide in the absence of a modulator compound; contacting the cell with the modulator compound and determining the biological activity of the expressed LSI-1 polypeptide in the presence of the modulator compound. In such a method, a difference between the activity of the LSI-1 polypeptide in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.

Essentially any chemical compound can be employed as a potential modulator or ligand in the assays according to the present invention. Compounds tested as calpain modulators can be any small chemical compound, or biological entity (e.g., protein, sugar, nucleic acid, lipid). Test compounds will typically be small chemical molecules and peptides. Generally, the compounds used as potential modulators can be dissolved in aqueous or organic (e.g., DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source. Assays are typically run in parallel, for example, in microtiter formats on microtiter plates in robotic assays. There are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), for example. Also, compounds may be synthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned for the detection of modulators of the novel LSI-1 polynucleotides and polypeptides described herein. Such high throughput screening methods typically involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (e.g., ligand or modulator compounds). Such combinatorial chemical libraries or ligand libraries are then screened in one or more assays to identify those library members (e.g., particular chemical species or subclasses) that display a desired characteristic activity. The compounds so identified can serve as conventional lead compounds, or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated either by chemical synthesis or biological synthesis, by combining a number of chemical building blocks (i.e., reagents such as amino acids). As an example, a linear combinatorial library, e.g., a polypeptide or peptide library, is formed by combining a set of chemical building blocks in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide or peptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries is well known to those having skill in the pertinent art. Combinatorial libraries include, without limitation, peptide libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; and Houghton et al., 1991, Nature, 354:84-88). Other chemistries for generating chemical diversity libraries can also be used. Nonlimiting examples of chemical diversity library chemistries include, peptoids (PCT Publication No. WO 91/019735), encoded peptides (PCT Publication No. WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagihara et al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of small compound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661), oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidyl phosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) and PCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996, Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organic molecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a large number of combinatorial libraries are commercially available (e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the cell or tissue expressing an ion channel is attached to a solid phase substrate. In such high throughput assays, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to perform a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; thus, for example, assay screens for up to about 6,000-20,000 different compounds are possible using the described integrated systems.

In another of its aspects, the present invention encompasses screening and small molecule (e.g., drug) detection assays which involve the detection or identification of small molecules that can bind to a given protein, i.e., a LSI-1 polypeptide or peptide. Particularly preferred are assays suitable for high throughput screening methodologies.

In such binding-based detection, identification, or screening assays, a functional assay is not typically required. All that is needed is a target protein, preferably substantially purified, and a library or panel of compounds (e.g., ligands, drugs, small molecules) or biological entities to be screened or assayed for binding to the protein target. Preferably, most small molecules that bind to the target protein will modulate activity in some manner, due to preferential, higher affinity binding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assay allows the detection of small molecules (e.g., drugs, ligands) that bind to expressed, and preferably purified, ion channel polypeptide based on affinity of binding determinations by analyzing thermal unfolding curves of protein-drug or ligand complexes. The drugs or binding molecules determined by this technique can be further assayed, if desired, by methods, such as those described herein, to determine if the molecules affect or modulate function or activity of the target protein.

To purify a LSI-1 polypeptide or peptide to measure a biological binding or ligand binding activity, the source may be a whole cell lysate that can be prepared by successive freeze-thaw cycles (e.g., one to three) in the presence of standard protease inhibitors. The LSI-1 polypeptide may be partially or completely purified by standard protein purification methods, e.g., affinity chromatography using specific antibody described infra, or by ligands specific for an epitope tag engineered into the recombinant LSI-1 polypeptide molecule, also as described herein. Binding activity can then be measured as described.

Compounds which are identified according to the methods provided herein, and which modulate or regulate the biological activity or physiology of the LSI-1 polypeptides according to the present invention are a preferred embodiment of this invention. It is contemplated that such modulatory compounds may be employed in treatment and therapeutic methods for treating a condition that is mediated by the novel LSI-1 polypeptides by administering to an individual in need of such treatment a therapeutically effective amount of the compound identified by the methods described herein.

In addition, the present invention provides methods for treating an individual in need of such treatment for a disease, disorder, or condition that is mediated by the LSI-1 polypeptides of the invention, comprising administering to the individual a therapeutically effective amount of the LSI-1-modulating compound identified by a method provided herein.

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:1, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone. In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoR1 site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bemoist and Chambon, Nature, 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region are most preferred.

Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al, Science, 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs corresponding to the polynucleotides of the invention, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA corresponding to the polynucleotides of the invention; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the polynucleotides of the invention in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/or diagnose the diseases described herein.

Thus, the invention provides a method of treating or preventing diseases, disorders, and/or conditions, including but not limited to the diseases, disorders, and/or conditions listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention. invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.

Biotic Associations

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations with other organisms. Such associations may be symbiotic, nonsymbiotic, endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. In general, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability to form biotic associations with any member of the fungal, bacterial, lichen, mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom, phylums, families, classes, genuses, and/or species.

The mechanism by which a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the host organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations is variable, though may include, modulating osmolarity to desirable levels for the symbiont, modulating pH to desirable levels for the symbiont, modulating secretions of organic acids, modulating the secretion of specific proteins, phenolic compounds, nutrients, or the increased expression of a protein required for host-biotic organisms interactions (e.g., a receptor, ligand, etc.). Additional mechanisms are known in the art and are encompassed by the invention (see, for example, “Microbial Signalling and Communication”, eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, Cambridge University Press, Cambridge, (1999); which is hereby incorporated herein by reference).

In an alternative embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may decrease the host organisms ability to form biotic associations with another organism, either directly or indirectly. The mechanism by which a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may decrease the host organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations with another organism is variable, though may include, modulating osmolarity to undesirable levels, modulating pH to undesirable levels, modulating secretions of organic acids, modulating the secretion of specific proteins, phenolic compounds, nutrients, or the decreased expression of a protein required for host-biotic organisms interactions (e.g., a receptor, ligand, etc.). Additional mechanisms are known in the art and are encompassed by the invention (see, for example, “Microbial Signalling and Communication”, eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, Cambridge University Press, Cambridge, (1999); which is hereby incorporated herein by reference).

The hosts ability to maintain biotic associations with a particular pathogen has significant implications for the overall health and fitness of the host. For example, human hosts have symbiosis with enteric bacteria in their gastrointestinal tracts, particularly in the small and large intestine. In fact, bacteria counts in feces of the distal colon often approach 10¹² per milliliter of feces. Examples of bowel flora in the gastrointestinal tract are members of the Enterobacteriaceae, Bacteriodes, in addition to a-hemolytic streptococci, E. coli, Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli, and yeasts. Such bacteria, among other things, assist the host in the assimilation of nutrients by breaking down food stuffs not typically broken down by the hosts digestive system, particularly in the hosts bowel. Therefore, increasing the hosts ability to maintain such a biotic association would help assure proper nutrition for the host.

Aberrations in the enteric bacterial population of mammals, particularly humans, has been associated with the following disorders: diarrhea, ileus, chronic inflammatory disease, bowel obstruction, duodenal diverticula, biliary calculous disease, and malnutrition. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention are useful for treating, detecting, diagnosing, prognosing, and/or ameliorating, either directly or indirectly, and of the above mentioned diseases and/or disorders associated with aberrant enteric flora population.

The composition of the intestinal flora, for example, is based upon a variety of factors, which include, but are not limited to, the age, race, diet, malnutrition, gastric acidity, bile salt excretion, gut motility, and immune mechanisms. As a result, the polynucleotides and polypeptides, including agonists, antagonists, and fragments thereof, may modulate the ability of a host to form biotic associations by affecting, directly or indirectly, at least one or more of these factors.

Although the predominate intestinal flora comprises anaerobic organisms, an underlying percentage represents aerobes (e.g., E. coli). This is significant as such aerobes rapidly become the predominate organisms in intraabdominal infections—effectively becoming opportunistic early in infection pathogenesis. As a result, there is an intrinsic need to control aerobe populations, particularly for immune compromised individuals.

In a preferred embodiment, a polynucleotides and polypeptides, including agonists, antagonists, and fragments thereof, are useful for inhibiting biotic associations with specific enteric symbiont organisms in an effort to control the population of such organisms.

Biotic associations occur not only in the gastrointestinal tract, but also on an in the integument. As opposed to the gastrointestinal flora, the cutaneous flora is comprised almost equally with aerobic and anaerobic organisms. Examples of cutaneous flora are members of the gram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci, micrococcus, M. sedentarius), gram-positive bacilli (e.g., Corynebacterium species, C. minutissimum, Brevibacterium species, Propoionibacterium species, P. acnes), gram-negative bacilli (e.g., Acinebacter species), and fungi (Pityrosporum orbiculare). The relatively low number of flora associated with the integument is based upon the inability of many organisms to adhere to the skin. The organisms referenced above have acquired this unique ability. Therefore, the polynucleotides and polypeptides of the present invention may have uses which include modulating the population of the cutaneous flora, either directly or indirectly.

Aberrations in the cutaneous flora are associated with a number of significant diseases and/or disorders, which include, but are not limited to the following: impetigo, ecthyma, blistering distal dactulitis, pustules, folliculitis, cutaneous abscesses, pitted keratolysis, trichomycosis axcillaris, dermatophytosis complex, axillary odor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheic dermititis, and Pityrosporum folliculitis, to name a few. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention are useful for treating, detecting, diagnosing, prognosing, and/or ameliorating, either directly or indirectly, and of the above mentioned diseases and/or disorders associated with aberrant cutaneous flora population.

Additional biotic associations, including diseases and disorders associated with the aberrant growth of such associations, are known in the art and are encompassed by the invention. See, for example, “Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G., Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia, (1998); which is hereby incorporated herein by reference).

Pheromones

In another embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability to synthesize and/or release a pheromone. Such a pheromone may, for example, alter the organisms behavior and/or metabolism.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may modulate the biosynthesis and/or release of pheromones, the organisms ability to respond to pheromones (e.g., behaviorally, and/or metabolically), and/or the organisms ability to detect pheromones. Preferably, any of the pheromones, and/or volatiles released from the organism, or induced, by a polynucleotide or polypeptide and/or agonist or antagonist of the invention have behavioral effects the organism.

Other Activities

The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat, prevent, and/or diagnose neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive diseases, disorders, and/or conditions), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to increase the efficacy of a pharmaceutical composition, either directly or indirectly. Such a use may be administered in simultaneous conjunction with said pharmaceutical, or separately through either the same or different route of administration (e.g., intravenous for the polynucleotide or polypeptide of the present invention, and orally for the pharmaceutical, among others described herein.).

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to prepare individuals for extraterrestrial travel, low gravity environments, prolonged exposure to extraterrestrial radiation levels, low oxygen levels, reduction of metabolic activity, exposure to extraterrestrial pathogens, etc. Such a use may be administered either prior to an extraterrestrial event, during an extraterrestrial event, or both. Moreover, such a use may result in a number of beneficial changes in the recipient, such as, for example, any one of the following, non-limiting, effects: an increased level of hematopoietic cells, particularly red blood cells which would aid the recipient in coping with low oxygen levels; an increased level of B-cells, T-cells, antigen presenting cells, and/or macrophages, which would aid the recipient in coping with exposure to extraterrestrial pathogens, for example; a temporary (i.e., reversible) inhibition of hematopoietic cell production which would aid the recipient in coping with exposure to extraterrestrial radiation levels; increase and/or stability of bone mass which would aid the recipient in coping with low gravity environments; and/or decreased metabolism which would effectively facilitate the recipients ability to prolong their extraterrestrial travel by any one of the following, non-limiting means: (i) aid the recipient by decreasing their basal daily energy requirements; (ii) effectively lower the level of oxidative and/or metabolic stress in recipient (i.e., to enable recipient to cope with increased extraterrestial radiation levels by decreasing the level of internal oxidative/metabolic damage acquired during normal basal energy requirements; and/or (iii) enabling recipient to subsist at a lower metabolic temperature (i.e., cryogenic, and/or sub-cryogenic environment).

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:1 wherein X is any integer as defined in Table I.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:1 in the range of positions beginning with the nucleotide at about the position of the “5′ NT of Start Codon of ORF” and ending with the nucleotide at about the position of the “3′ NT of ORF” as defined for SEQ ID NO:1 in Table I.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:1.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:1.

A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:1 beginning with the nucleotide at about the position of the “5′ NT of ORF” and ending with the nucleotide at about the position of the “3′ NT of ORF” as defined for SEQ ID NO:1 in Table I.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:1.

Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.

Also preferred is a composition of matter comprising a DNA molecule which comprises a cDNA clone identified by a cDNA Clone Identifier in Table I, which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table I for said cDNA Clone Identifier.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a cDNA clone identified by a cDNA Clone Identifier in Table I, which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table I.

Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said cDNA clone.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by said cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by said cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by said cDNA clone.

A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:1 wherein X is any integer as defined in Table I; and a nucleotide sequence encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.

Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:1 wherein X is any integer as defined in Table I; and a nucleotide sequence encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a protein identified in Table I, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:1 wherein X is any integer as defined in Table I; and a nucleotide sequence encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:1 wherein X is any integer as defined in Table I; and a nucleotide sequence encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I.

Also preferred is a polypeptide, wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO:2 in the range of positions “Total AA of the Open Reading Frame (ORF)” as set forth for SEQ ID NO:2 in Table I.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:2.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:2.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:2.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of the protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of the protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I; which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.

Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.

Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.

Also preferred is a method for diagnosing a pathological condition associated with an organism with abnormal structure or expression of a gene encoding a protein identified in Table I, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule(s) into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is an integer set forth in Table I and said position of the “Total AA of ORF” of SEQ ID NO:2 is defined in Table I; and an amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I. The isolated polypeptide produced by this method is also preferred.

Also preferred is a method of treatment of an individual in need of an increased level of a protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

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EXAMPLES DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 Bioinformatics Analysis

To search for novel protease inhibitors, a Hidden-Markov Model (HMM) of serine protease inhibitors SERPIN (obtained from the Pfam database in Sanger center) (Bateman et. al., 2000) was used to search against human genomic sequence database using the computer program GENEWISEDB. Genomic sequences that were found to have a GENEWISEDB matching score of more than 15 against SERPIN HMM model were selected for further analysis. Genomic sequence contained in BAC (bacteria artificial chromosome) AL132708 was found to contain putative exon sequences that are similar to serpins. The portion of sequence from AL132708 that matched Peptidase_M22 HMM profile was extracted and back-searched against non-redundant protein database using BLASTX program (Altschul et. al., 1990). The most similar protein sequence was used as a template to predict more exons from AL132708 using GENEWISEDB program (Birney and Durbin, 2000). The final predicted exons were assembled and a full-length clone of gene LSI-01 was obtained using the predicted exon sequences. The complete protein sequence of LSI-01 was found to have significant sequence homology with a family of known protease inhibitors. A peptide sequence VSFNRTFLMMI fitting the conserved peptide signature pattern of serpins [LIVMFY]-x-[LIVMFYAC]-[DNQ]-[RKHQS]-[PST]-F-[LIVMFY]-[LIVMFYC]-x-[LIVMFAH] is found in the protein sequence of LSI-01 (Hofmann et. al., 1999).

Proteins in the SERPIN family are secreted proteins. AL132708 contains a strong signal sequence at the NH₂ terminal (see FIGS. 1A-B), suggesting that LSI-01 is also a secreted protein. Protein threading and molecular modeling of LSI-01 suggest that LSI-01 have a structure folding of serine protease inhibitor, with RS as putative bait sequence. Based on sequence, structure and known SERPIN signature sequences, the novel LSI-01 is a novel human serine protease inhibitor.

Example 2 Method for Constructing a Size Fractionated Brain cDNA Library

Brain poly A+RNA was purchased from Clontech and converted into double stranded cDNA using the SuperScript™ Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) except that no radioisotope was incorporated in either of the cDNA synthesis steps and that the cDNA was fractionated by HPLC. This was accomplished on a TransGenomics HPLC system equipped with a size exclusion column (TosoHass) with dimensions of 7.8 mm×30 cm and a particle size of 10 □m. Tris buffered saline was used as the mobile phase and the column was run at a flow rate of 0.5 mL/min.

The resulting chromatograms were analyzed to determine which fractions should be pooled to obtain the largest cDNA's; generally fractions that eluted in the range of 12 to 15 minutes were pooled. The cDNA was precipitated prior to ligation into the Sal I/Not I sites in the pSport vector supplied with the kit. Using a combination of PCR with primers to the ends of the vector and Sal I/Not I restriction enzyme digestion of mini-prep DNA, it was determined that the average insert size of the library was greater the 3.5 Kb. The overall complexity of the library was greater that 10⁷ independent clones. The library was amplified in semi-solid agar for 2 days at 30° C. An aliquot (200 microliters) of the amplified library was inoculated into a 200 ml culture for single-stranded DNA isolation by super-infection with a f1 helper phage. After overnight growth, the released phage particles with precipitated with PEG and the DNA isolated with proteinase K, SDS and phenol extractions. The single stranded circular DNA was concentrated by ethanol precipitation and used for the cDNA capture experiments.

Example 3 Cloning of the Novel Human LSI-01 Serpin

Using the predict exon genomic sequence from bac AL132708, an antisense 80 bp oligonucleotide with biotin on the 5′ end was designed with the following sequence; 5′bGGCACTTCCCATCTTCAAGGTCAGGTCTTTGCT (SEQ ID NO:18) GGGAACAGTCAGTGAGTGAACCAGGTGCTGGAAGCC CTG-3′

One microliter (one hundred and fifty nanograms) of the biotinylated oligonucleotide was added to six microliters (six micrograms) of a mixture of single-stranded covalently closed circular liver and spleen cDNA libraries (These libraries are commercially available from Life Technologies, Rockville, Md.) and seven microliters of 100% formamide in a 0.5 ml PCR tube. The mixture was heated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of 2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA library mixture and incubated at 42° C. for 26 hours. Hybrids between the biotinylated oligonucleotide and the circular cDNA were isolated by diluting the hybridization mixture to 220 microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magnetic beads. This solution was incubated at 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. The beads were separated from the solution with a magnet and the beads washed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyated oligonucleotide/streptavidin magnetic bead complex by adding 50 microliters of 0.1 N NaOH and incubating at room temperature for 10 mins. Six microliters of 3 M Sodium Acetate was added along with 15 micrograms of glycogen and the solution ethanol precipitated with 120 microliters of 100% ethanol. The DNA was resuspended in 12 microliters of TE (10 mM Tris-HCl, pH 8.0), 1 mM EDTA, pH 8.0). The single stranded cDNA was converted into double strands in a thermal cycler by mixing 5 microliters of the captured DNA with 1.5 microliters 10 micromolar standard SP6 primer (homologous to a sequence on the cDNA cloning vector) and 1.5 microliters of 10× PCR buffer. The mixture was heated to 95° C. for 20 seconds then ramped down to 59° C. At this time 15 microliters of a repair mix, that was preheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs (1.25 mM each), 1.5 microliters of 10× PCR buffer, 9.25 microliters of water, and 0.25 microliters of Taq polymerase). The solution was ramped back to 73° C. and incubated for 23 mins. The repaired DNA was ethanol precipitate and resuspended in 10 microliters of TE. Two microliters were electroporated in E. coli DH12S cells and resulting colonies were screen by PCR, using a primer pair designed from the genomic exonic sequence to identify the proper cDNAs.

Oligonucleotides used to identity the cDNA by PCR. AL132708-L1 GCCTCTGTGCTCCAATCTACT (SEQ ID NO:19) AL132708-R1 GCTGCAGCTCCTTCTTGAC (SEQ ID NO:20)

Those cDNA clones that were positive by PCR had the inserts sized and two clones were chosen for DNA sequencing. Both clones had identical sequence.

The full-length nucleotide sequence and the encoded polypeptide for LSI-01 is shown in FIGS. 1A-B. The sequence was analyzed and plotted in a hydrophobicity plot showing a putative signal sequence at the NH₂ terminal (see FIG. 4).

Example 4 Expression Profiling of the Novel Human LSI-01 Serpin

The same PCR primer pair that was used to identify the novel LSI-01 cDNA clones (SEQ ID NO:18 and 19) was used to measure the steady state levels of mRNA by quantitative PCR. Briefly, first strand cDNA was made from commercially available mRNA. The relative amount of cDNA used in each assay was determined by performing a parallel experiment using a primer pair for a gene expressed in equal amounts in all tissues, cyclophilin. The cyclophilin primer pair detected small variations in the amount of cDNA in each sample and these data were used for normalization of the data obtained with the primer pair for the novel LSI-01. The PCR data was converted into a relative assessment of the difference in transcript abundance amongst the tissues tested and the data is presented in FIG. 5. Transcripts corresponding to LSI-01 were expressed highly in the lymph node, and to a lesser extent in thumus, small intestine, and spleen.

Example 5 Method of Measuring the Serine Protease Inhibitory Activity of LSI-01 Polypeptides

Serpin (serine protease inhibitory) activity of the LSI-01 polypeptide may be measured by following the inhibition of proteolytic activity in cells, tissues, and/or in in vitro assays. The LSI-01 serpin may be incubated with one or more proteases (preferably Arg-specific proteases provided below and elsewhere herein) for different times and with varying serpin concentrations. Residual protease activity could then be measured according to any appropriate means known in the art. As discussed elsewhere herein, serpin activity results in the inhibition of protease activity. Likewise, serpin inhibitors or antagonists may be capable of preventing the loss of such protease activity.

In vitro assays for measuring protease activity using synthetic peptide fluorescent, spectrophotometric and fluorescence resonance transfer assays are well described in the art, either through the use of single substrates (see below for examples), or as part of substrate libraries (Backes et al., 2000).

Non-limiting examples of Arg-specific proteases include the coagulation proteases (thrombin, FXa, and TF:FVIIa), the fibrinolytic enzymes (tPA, uPA and plasmin), and the inflammatory enzyme tryptase. Other Arg-specific proteases may be known in the art and are encompassed by the present invention. Assays for thrombin, plasmin, uPA, tPA, and tryptase are well described in the art (Balasubramanian et al., 1993; Combrink et al., 1998).

An example of a spectrophotometric protease assay is the Factor Xa assay. Briefly, human FXa (Calbiochem #233526) enzymatic activity is measured in a buffer containing 0.145 M NaCl, 0.005 M KCl, 1 mg/ml Polyethylene Glycol (PEG-8000), 0.030 M HEPES (pH 7.4) using 96-well microtiter plates (Nunc Immuno #439454). The enzyme is incubated with the serpin at room temperature for varying amounts of time prior to starting the reaction with 100 μM S-2222 (phenyl-Ile-Glu-Gly-Arg-pNA, K_(m)=137 μM). The Km for this, and other substrates, may be determined experimentally by measuring the enzyme activity at different substrate concentrations and curve fitting the data using Kaleidagraph V. Time-dependent optical density change may be followed at 405 nm using a kinetic microplate reader (Molecular Devices UVmax) at room temperature. Enzyme activity in the presence of serpin (LSI-01) may be expressed as fraction of control and curve fit to pre-incubation time and serpin concentration to determine inhibitory parameters.

An example of a fluorescence assay which may be used for the present invention is the Factor VIIa assay. Briefly, the Factor VIIa assay is measured in the presence of human recombinant tissue factor (INNOVIN from Dade Behring Cat.# B4212-100). Human Factor VIIa may be obtained from Enzyme Research Labs (Cat.# HFVIIA 1640). Enzymatic activity could be measured in a buffer containing 150 mM NaCl, 5 mM CaCl₂, 1 mM CHAPS and 1 mg/ml PEG 6000 (pH 7.4) with 1 nM FVIIa and 100 μM D-Ile-Pro-Arg-AFC (Enzyme Systems Products, Km>200 μM) 0.66% DMSO. The assay (302 μl total volume) may be incubated at room temperature for 2 hr prior to reading fluorometric signal (Ex 405/Em 535) using a Victor 2 (Wallac) fluorescent plate reader.

In addition to the methods described above, protease activity (and therefore serpin activity) can be measured using fluorescent resonance energy transfer (FRET with Quencher —P_(n)—P₃—P₂—P₁— —P₁′—P₂′— Fluorophore), fluorescent peptide bound to beads (Fluorophore —P_(n)—P₃—P₂—P₁— —P₁′—P₂′Bead), dye-protein substrates and serpin-protease gel shifts. All of which are well known to those skilled in the art.

Additional assays, in addition to, assay methods are known in the art and are encompassed by the present inventoin. See, for example, Backes B J, Harris J L, Leonetti F, Craik C S, Ellman J A. Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat Biotechnol. 18:187-93 (2000); Balasubramanian, N., St. Laurent, D. R., Federici, M. E., Meanwell, N. A., Wright, J. J., Schumacher, W. A., and Seiler, S. M. Active site-directed synthetic thrombin inhibitors: synthesis, in vitro and in vivo activity profile of BMY 44621 and analogs. an examination of the role of the amino group in the D-Phe-Pro-Arg-H series. J. Med. Chem. 36:300-303 (1993); and Combrink, K. D., Gülgeze, H. B., Meanwell, N. A., Pearce, B. C. Zulan, P., Bisacchi, G. S., Roberts, D. G. M., Stanley, P. Seiler, S. M. Novel 1,2-Benzisothiazol-3-one-1,1-dioxide Inhibitors of Human Mast Cell Tryptase. J. Med. Chem. 41:4854-4860 (1998); which are hereby incorporated herein by reference in their entirety.

Additional methods of assessing LSI-01 serpin activity may be in reference to the methods disclosed in any of the publications referenced herein.

Example 6 Method of Screening for Compounds that Interact with the LSI-01 Polypeptide

The following assays are designed to identify compounds that bind to the LSI-01 polypeptide, bind to other cellular proteins that interact with the LSI-01 polypeptide, and to compounds that interfere with the interaction of the LSI-01 polypeptide with other cellular proteins.

Such compounds can include, but are not limited to, other cellular proteins. Specifically, such compounds can include, but are not limited to, peptides, such as, for example, soluble peptides, including, but not limited to Ig-tailed fusion peptides, comprising extracellular portions of LSI-01 polypeptide transmembrane receptors, and members of random peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghton, R. et al., 1991, Nature 354:84-86), made of D-and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate phosphopeptide libraries; see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′).sub.2 and FAb expression libary fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can be useful, for example, in elaborating the biological function of the LSI-01 polypeptide, and for ameliorating symptoms of tumor progression, for example. In instances, for example, whereby a tumor progression state or disorder results from a lower overall level of LSI-01 expression, LSI-01 polypeptide, and/or LSI-01 polypeptide activity in a cell involved in the tumor progression state or disorder, compounds that interact with the LSI-01 polypeptide can include ones which accentuate or amplify the activity of the bound LSI-01 polypeptide. Such compounds would bring about an effective increase in the level of LSI-01 polypeptide activity, thus ameliorating symptoms of the tumor progression disorder or state. In instances whereby mutations within the LSI-01 polypeptide cause aberrant LSI-01 polypeptides to be made which have a deleterious effect that leads to tumor progression, compounds that bind LSI-01 polypeptide can be identified that inhibit the activity of the bound LSI-01 polypeptide. Assays for testing the effectiveness of such compounds are known in the art and discussed, elsewhere herein.

Example 7 Method of Screening, In Vitro, Compounds that Bind to the LSI-01 Polypeptide

In vitro systems can be designed to identify compounds capable of binding the LSI-01 polypeptide of the invention. Compounds identified can be useful, for example, in modulating the activity of wild type and/or mutant LSI-01 polypeptide, preferably mutant LSI-01 polypeptide, can be useful in elaborating the biological function of the LSI-01 polypeptide, can be utilized in screens for identifying compounds that disrupt normal LSI-01 polypeptide interactions, or can in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to the LSI-01 polypeptide involves preparing a reaction mixture of the LSI-01 polypeptide and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring LSI-01 polypeptide or the test substance onto a solid phase and detecting LSI-01 polypeptide test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the LSI-01 polypeptide can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly.

In practice, microtitre plates can conveniently be utilized as the solid phase. The anchored component can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for LSI-01 polypeptide or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

Example 8 Method of Identifying Compounds that Interfere with LSI-01 Polypeptide/Cellular Product Interaction

The LSI-01 polypeptide of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. Such macromolecules include, but are not limited to, nucleic acid molecules and those products identified via methods such as those described, elsewhere herein. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partner(s)”. For the purpose of the present invention, “binding partner” may also encompass small molecule compounds, polysaccarides, lipids, and any other molecule or molecule type referenced herein. Compounds that disrupt such interactions can be useful in regulating the activity of the LSI-01 polypeptide, especially mutant LSI-01 polypeptide. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and the like described in elsewhere herein.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the LSI-01 polypeptide and its cellular or extracellular binding partner or partners involves preparing a reaction mixture containing the LSI-01 polypeptide, and the binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of LSI-01 polypeptide and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the LSI-01 polypeptide and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the LSI-01 polypeptide and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal LSI-01 polypeptide can also be compared to complex formation within reaction mixtures containing the test compound and mutant LSI-01 polypeptide. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal LSI-01 polypeptide.

The assay for compounds that interfere with the interaction of the LSI-01 polypeptide and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the LSI-01 polypeptide or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the LSI-01 polypeptide and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the LSI-01 polypeptide and interactive cellular or extracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the LSI-01 polypeptide or the interactive cellular or extracellular binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtitre plates are conveniently utilized. The anchored species can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished simply by coating the solid surface with a solution of the LSI-01 polypeptide or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the LSI-01 polypeptide and the interactive cellular or extracellular binding partner product is prepared in which either the LSI-01 polypeptide or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt LSI-01 polypeptide -cellular or extracellular binding partner interaction can be identified.

In a particular embodiment, the LSI-01 polypeptide can be prepared for immobilization using recombinant DNA techniques known in the art. For example, the LSI-01 polypeptide coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector such as pGEX-5X-1, in such a manner that its binding activity is maintained in the resulting fusion product. The interactive cellular or extracellular product can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above. This antibody can be labeled with the radioactive isotope.sup.125 I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST- LSI-01 polypeptide fusion product can be anchored to glutathione-agarose beads. The interactive cellular or extracellular binding partner product can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the LSI-01 polypeptide and the interactive cellular or extracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-LSI-01 polypeptide fusion product and the interactive cellular or extracellular binding partner product can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the binding partners are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the LSI-01 polypeptide product and the interactive cellular or extracellular binding partner (in case where the binding partner is a product), in place of one or both of the full length products.

Any number of methods routinely practiced in the art can be used to identify and isolate the protein's binding site. These methods include, but are not limited to, mutagenesis of one of the genes encoding one of the products and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can be selected. Sequence analysis of the genes encoding the respective products will reveal the mutations that correspond to the region of the product involved in interactive binding. Alternatively, one product can be anchored to a solid surface using methods described in this Section above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain can remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the cellular or extracellular binding partner product is obtained, short gene segments can be engineered to express peptide fragments of the product, which can then be tested for binding activity and purified or synthesized.

Example 9 Isolation of a Specific Clone from the Deposited Sample

The deposited material in the sample assigned the ATCC Deposit Number cited in Table I for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table I. Typically, each ATCC deposit sample cited in Table I comprises a mixture of approximately equal amounts (by weight) of about 1-10 plasmid DNAs, each containing a different cDNA clone and/or partial cDNA clone; but such a deposit sample may include plasmids for more or less than 2 cDNA clones.

Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNA(s) cited for that clone in Table I. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to SEQ ID NO:1.

Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with 32P-(-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ ID NO:1 (i.e., within the region of SEQ ID NO:1 bounded by the 5′ NT and the 3′ NT of the clone defined in Table I) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.

The polynucleotide(s) of the present invention, the polynucleotide encoding the polypeptide of the present invention, or the polypeptide encoded by the deposited clone may represent partial, or incomplete versions of the complete coding region (i.e., full-length gene). Several methods are known in the art for the identification of the 5′ or 3′ non-coding and/or coding portions of a gene which may not be present in the deposited clone. The methods that follow are exemplary and should not be construed as limiting the scope of the invention. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols that are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full-length gene.

This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA that may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene. Moreover, it may be advantageous to optimize the RACE protocol to increase the probability of isolating additional 5′ or 3′ coding or non-coding sequences. Various methods of optimizing a RACE protocol are known in the art, though a detailed description summarizing these methods can be found in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

An alternative method for carrying out 5′ or 3′ RACE for the identification of coding or non-coding sequences is provided by Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). Briefly, a cDNA clone missing either the 5′ or 3′ end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start of translation, therefor. The following briefly describes a modification of this original 5′ RACE procedure. Poly A+ or total RNAs reverse transcribed with Superscript II (Gibco/BRL) and an antisense or I complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or SalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5′ ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32(1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNA library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to Obtain Full Length Genes

Once a gene of interest is identified, several methods are available for the identification of the 5′ or 3′ portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5′ and 3′RACE. While the full-length gene may be present in the library and can be identified by probing, a useful method for generating the 5′ or 3′ end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5′RACE is available for generating the missing 5′ end of a desired full-length gene. (This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7): 1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably 30 containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5′ portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the apoptosis related of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the relevant apoptosis related.

Example 10 Tissue Distribution of Polypeptide

Tissue distribution of mRNA expression of polynucleotides of the present invention is determined using protocols for Northern blot analysis, described by, among others, Sambrook et al. For example, a cDNA probe produced by the method described in Example 9 is labeled with p32 using the rediprime™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using CHROMA SPINO-100 column (Clontech Laboratories, Inc.) according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various tissues for mRNA expression.

Tissue Northern blots containing the bound mRNA of various tissues are examined with the labeled probe using ExpressHyb™ hybridization solution (Clonetech according to manufacturers protocol number PT1190-1. Northern blots can be produced using various protocols well known in the art (e.g., Sambrook et al). Following hybridization and washing, the blots are mounted and exposed to film at −70C overnight, and the films developed according to standard procedures.

Example 11 Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO:1. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds,95 degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C. Mammalian DNA, preferably human DNA, is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions are analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 12 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 9, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, that expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000 Xg). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni—NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni—NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni—NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C. or frozen at −80 degree C.

Example 13 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.

The cells are then lysed by passing the solution through a microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000 xg for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000 xg centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C. overnight to allow further GuHCl extraction.

Following high speed centrifugation (30,000 xg) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C. without mixing for 12 hours prior to further purification steps.

To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Coomassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.

Example 14 Cloning and Expression of a Polypeptide in a Baculovirus Expression System

In this example, the plasmid shuttle vector pAc373 is used to insert a polynucleotide into a baculovirus to express a polypeptide. A typical baculovirus expression vector contains the strong polyhedrin promoter of the Autographa califomica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites, which may include, for example BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV4038 ) is often used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above, such as pVL941 and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 9, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites at the 5′ end of the primers in order to clone the amplified product into the expression vector. Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified elsewhere herein (if applicable), is amplified using the PCR protocol described in Example 9. If the naturally occurring signal sequence is used to produce the protein, the vector used does not need a second signal peptide. Alternatively, the vector can be modified to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

Five ug of a plasmid containing the polynucleotide is co-transformed with 1.0 ug of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C. for four days.

After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.

Example 15 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transformation with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transformed cells.

The transformed gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

A polynucleotide of the present invention is amplified according to the protocol outlined in herein. If the naturally occurring signal sequence is used to produce the protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.) The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used for transformation. Five μg of an expression plasmid is cotransformed with 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

Example 16 Protein Fusions

The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example described herein; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the half-life time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.

The naturally occurring signal sequence may be used to produce the protein (if applicable). Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat. No. 6,066,781, supra.)

Human IgG Fc region:      GGGATCCGGAGCCCAAATCTTCTGACAAAAC (SEQ ID NO:32) TCACACATGCCCACCGTGCCCAGCACCTGAATTCGA GGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACC CAAGGACACCCTCATGATCTCCCGGACTCCTGAGGT CACATGCGTGGTGGTGGACGTAAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG GGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGT GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC CTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACG GCCGCGACTCTAGAGGAT

Example 17 Production of an Antibody from a Polypeptide

The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of the protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Alternatively, protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

Moreover, in another preferred method, the antibodies directed against the polypeptides of the present invention may be produced in plants. Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and 6,080,560, which are hereby incorporated in their entirety herein. The methods not only describe methods of expressing antibodies, but also the means of assembling foreign multimeric proteins in plants (i.e., antibodies, etc,), and the subsequent secretion of such antibodies from the plant.

Example 18 Method of Creating N— and C-terminal Deletion Mutants Corresponding to the LSI-01 Polypeptide of the Present Invention

As described elsewhere herein, the present invention encompasses the creation of N— and C-terminal deletion mutants, in addition to any combination of N— and C-terminal deletions thereof, corresponding to the LSI-01 polypeptide of the present invention. A number of methods are available to one skilled in the art for creating such mutants. Such methods may include a combination of PCR amplification and gene cloning methodology. Although one of skill in the art of molecular biology, through the use of the teachings provided or referenced herein, and/or otherwise known in the art as standard methods, could readily create each deletion mutant of the present invention, exemplary methods are described below.

Briefly, using the isolated cDNA clone encoding the full-length LSI-01 polypeptide sequence (as described in Example 9, for example), appropriate primers of about 15-25 nucleotides derived from the desired 5′ and 3′ positions of SEQ ID NO:1 may be designed to PCR amplify, and subsequently clone, the intended N— and/or C-terminal deletion mutant. Such primers could comprise, for example, an inititation and stop codon for the 5′ and 3′ primer, respectively. Such primers may also comprise restriction sites to facilitate cloning of the deletion mutant post amplification. Moreover, the primers may comprise additional sequences, such as, for example, flag-tag sequences, kozac sequences, or other sequences discussed and/or referenced herein.

For example, in the case of the V39 to S435 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: 5′ 5′-GCAGCA GCGGCCGC GTGTCCCCGG (SEQ ID NO:34) Primer CCAATGCCCCCAGTG-3′     NotI 3′ 5′-GCAGCA GTCGAC GGATTTAGTGGG (SEQ ID NO:35) Primer ATTTTCCACTTTC-3′     SalI

For example, in the case of the M1 to T404 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: 5′ 5′-GCAGCA GCGGCCGC ATGCAAGGAC (SEQ ID NO:36) Primer AGGGCAGGAGAAGAG-3′     NotI 3′ 5′-GCAGCA GTCGAC TGACATCCAGCA (SEQ ID NO:37) Primer CAGCCTTGTGGG-3′     SalI

Representative PCR amplification conditions are provided below, although the skilled artisan would appreciate that other conditions may be required for efficient amplification. A 100 ul PCR reaction mixture may be prepared using long of the template DNA (cDNA clone of LSI-01), 200 uM 4dNTPs, 1 uM primers, 0.25U Taq DNA polymerase (PE), and standard Taq DNA polymerase buffer. Typical PCR cycling condition are as follows: 20-25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2 min, 72 degrees 1 cycle: 10 min, 72 degrees

After the final extension step of PCR, 5U Klenow Fragment may be added and incubated for 15 min at 30 degrees.

Upon digestion of the fragment with the NotI and SalI restriction enzymes, the fragment could be cloned into an appropriate expression and/or cloning vector which has been similarly digested (e.g., pSportl, among others). The skilled artisan would appreciate that other plasmids could be equally substituted, and may be desirable in certain circumstances. The digested fragment and vector are then ligated using a DNA ligase, and then used to transform competent E. coli cells using methods provided herein and/or otherwise known in the art.

The 5′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the LSI-01 gene (SEQ ID NO:1), and ‘X’ is equal to the most N-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 5′ primer, while the second term will provide the end 3′ nucleotide position of the 5′ primer corresponding to sense strand of SEQ ID NO:1. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 5′ primer may be desired in certain circumstances (e.g., kozac sequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))-25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the LSI-01 gene (SEQ ID NO:1), and ‘X’ is equal to the most C-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 3′ primer, while the second term will provide the end 3′ nucleotide position of the 3′ primer corresponding to the anti-sense strand of SEQ ID NO:1. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 3′ primer may be desired in certain circumstances (e.g., stop codon sequences, etc.). The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.

The same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any C-terminal deletion mutant of the present invention. Moreover, the same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any combination of N-terminal and C-terminal deletion mutant of the present invention. The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.

Example 19 Regulation of Protein Expression Via Controlled Aggregation in the Endoplasmic Reticulum

As described more particularly herein, proteins regulate diverse cellular processes in higher organisms, ranging from rapid metabolic changes to growth and differentiation. Increased production of specific proteins could be used to prevent certain diseases and/or disease states. Thus, the ability to modulate the expression of specific proteins in an organism would provide significant benefits.

Numerous methods have been developed to date for introducing foreign genes, either under the control of an inducible, constitutively active, or endogenous promoter, into organisms. Of particular interest are the inducible promoters (see, M. Gossen, et al., Proc. Natl. Acad. Sci. USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA, 91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346 (1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in addition to additional examples disclosed elsewhere herein). In one example, the gene for erthropoietin (Epo) was transferred into mice and primates under the control of a small molecule inducer for expression (e.g., tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512, (1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M. Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye et al., Science, 283:88 (1999). Although such systems enable efficient induction of the gene of interest in the organism upon addition of the inducing agent (i.e., tetracycline, rapamycin, etc.,), the levels of expression tend to peak at 24 hours and trail off to background levels after 4 to 14 days. Thus, controlled transient expression is virtually impossible using these systems, though such control would be desirable.

A new alternative method of controlling gene expression levels of a protein from a transgene (i.e., includes stable and transient transformants) has recently been elucidated (V. M. Rivera., et al., Science, 287:826-830, (2000)). This method does not control gene expression at the level of the mRNA like the aforementioned systems. Rather, the system controls the level of protein in an active secreted form. In the absence of the inducing agent, the protein aggregates in the ER and is not secreted. However, addition of the inducing agent results in dis-aggregation of the protein and the subsequent secretion from the ER. Such a system affords low basal secretion, rapid, high level secretion in the presence of the inducing agent, and rapid cessation of secretion upon removal of the inducing agent. In fact, protein secretion reached a maximum level within 30 minutes of induction, and a rapid cessation of secretion within 1 hour of removing the inducing agent. The method is also applicable for controlling the level of production for membrane proteins.

Detailed methods are presented in V. M. Rivera., et al., Science, 287:826-830, (2000)), briefly:

Fusion protein constructs are created using polynucleotide sequences of the present invention with one or more copies (preferably at least 2, 3, 4, or more) of a conditional aggregation domain (CAD) a domain that interacts with itself in a ligand-reversible manner (i.e., in the presence of an inducing agent) using molecular biology methods known in the art and discussed elsewhere herein. The CAD domain may be the mutant domain isolated from the human FKBP12 (Phe³⁶ to Met) protein (as disclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), or alternatively other proteins having domains with similar ligand-reversible, self-aggregation properties. As a principle of design the fusion protein vector would contain a furin cleavage sequence operably linked between the polynucleotides of the present invention and the CAD domains. Such a cleavage site would enable the proteolytic cleavage of the CAD domains from the polypeptide of the present invention subsequent to secretion from the ER and upon entry into the trans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)). Alternatively, the skilled artisan would recognize that any proteolytic cleavage sequence could be substituted for the furin sequence provided the substituted sequence is cleavable either endogenously (e.g., the furin sequence) or exogenously (e.g., post secretion, post purification, post production, etc.). The preferred sequence of each feature of the fusion protein construct, from the 5′ to 3′ direction with each feature being operably linked to the other, would be a promoter, signal sequence, “X” number of (CAD)x domains, the furin sequence (or other proteolytic sequence), and the coding sequence of the polypeptide of the present invention. The artisan would appreciate that the promotor and signal sequence, independent from the other, could be either the endogenous promotor or signal sequence of a polypeptide of the present invention, or alternatively, could be a heterologous signal sequence and promotor.

The specific methods described herein for controlling protein secretion levels through controlled ER aggregation are not meant to be limiting are would be generally applicable to any of the polynucleotides and polypeptides of the present invention, including variants, homologues, orthologs, and fragments therein.

Example 20 Alteration of Protein Glycosylation Sites to Enhance Characteristics of Polypeptides of the Invention

Many eukaryotic cell surface and proteins are post-translationally processed to incorporate N-linked and O-linked carbohydrates (Komfeld and Komfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion (Fieldler and Simons (1995) Cell, 81:309-312; Helenius (1994) Mol. Biol. Of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982) Cell, 37:417-427; Alexamnder and Elder (1984), Science, 226:1328-1330; and Flack et al., (1994), J. Biol. Chem., 269:14015-14020). In higher organisms, the nature and extent of glycosylation can markedly affect the circulating half-life and bio-availability of proteins by mechanisms involving receptor mediated uptake and clearance (Ashwell and Morrell, (1974), Adv. Enzymol., 41:99-128; Ashwell and Harford (1982), Ann. Rev. Biochem., 51:531-54). Receptor systems have been identified that are thought to play a major role in the clearance of serum proteins through recognition of various carbohydrate structures on the glycoproteins (Stockert (1995), Physiol. Rev., 75:591-609; Kery et al., (1992), Arch. Biochem. Biophys., 298:49-55). Thus, production strategies resulting in incomplete attachment of terminal sialic acid residues might provide a means of shortening the bioavailability and half-life of glycoproteins. Conversely, expression strategies resulting in saturation of terminal sialic acid attachment sites might lengthen protein bioavailability and half-life.

In the development of recombinant glycoproteins for use as pharmaceutical products, for example, it has been speculated that the pharmacodynamics of recombinant proteins can be modulated by the addition or deletion of glycosylation sites from a glycoproteins primary structure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53). However, studies have reported that the deletion of N-linked glycosylation sites often impairs intracellular transport and results in the intracellular accumulation of glycosylation site variants (Machamer and Rose (1988), J. Biol Chem., 263:5955-5960; Gallagher et al., (1992), J. Virology., 66:7136-7145; Collier et al., (1993), Biochem., 32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta, 1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). While glycosylation site variants of proteins can be expressed intracellularly, it has proved difficult to recover useful quantities from growth conditioned cell culture medium.

Moreover, it is unclear to what extent a glycosylation site in one species will be recognized by another species glycosylation machinery. Due to the importance of glycosylation in protein metabolism, particularly the secretion and/or expression of the protein, whether a glycosylation signal is recognized may profoundly determine a proteins ability to be expressed, either endogenously or recombinately, in another organism (i.e., expressing a human protein in E. coli, yeast, or viral organisms; or an E. coli, yeast, or viral protein in human, etc.). Thus, it may be desirable to add, delete, or modify a glycosylation site, and possibly add a glycosylation site of one species to a protein of another species to improve the proteins functional, bioprocess purification, and/or structural characteristics (e.g., a polypeptide of the present invention).

A number of methods may be employed to identify the location of glycosylation sites within a protein. One preferred method is to run the translated protein sequence through the PROSITE computer program (Swiss Institute of Bioinformatics). Once identified, the sites could be systematically deleted, or impaired, at the level of the DNA using mutagenesis methodology known in the art and available to the skilled artisan, Preferably using PCR-directed mutagenesis (See Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Similarly, glycosylation sites could be added, or modified at the level of the DNA using similar methods, preferably PCR methods (See, Maniatis, supra). The results of modifying the glycosylation sites for a particular protein (e.g., solubility, secretion potential, activity, aggregation, proteolytic resistance, etc.) could then be analyzed using methods know in the art.

The skilled artisan would acknowledge the existence of other computer algorithms capable of predicting the location of glycosylation sites within a protein. For example, the Motif computer program (Genetics Computer Group suite of programs) provides this function, as well.

Example 21 Method of Enhancing the Biological Activity/Functional Characteristics of Invention through Molecular Evolution

Although many of the most biologically active proteins known are highly effective for their specified function in an organism, they often possess characteristics that make them undesirable for transgenic, therapeutic, and/or industrial applications. Among these traits, a short physiological half-life is the most prominent problem, and is present either at the level of the protein, or the level of the proteins mRNA. The ability to extend the half-life, for example, would be particularly important for a proteins use in gene therapy, transgenic animal production, the bioprocess production and purification of the protein, and use of the protein as a chemical modulator among others. Therefore, there is a need to identify novel variants of isolated proteins possessing characteristics which enhance their application as a therapeutic for treating diseases of animal origin, in addition to the proteins applicability to common industrial and pharmaceutical applications.

Thus, one aspect of the present invention relates to the ability to enhance specific characteristics of invention through directed molecular evolution. Such an enhancement may, in a non-limiting example, benefit the inventions utility as an essential component in a kit, the inventions physical attributes such as its solubility, structure, or codon optimization, the inventions specific biological activity, including any associated enzymatic activity, the proteins enzyme kinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding activity, antagonist/inhibitory activity (including direct or indirect interaction), agonist activity (including direct or indirect interaction), the proteins antigenicity (e.g., where it would be desirable to either increase or decrease the antigenic potential of the protein), the immunogenicity of the protein, the ability of the protein to form dimers, trimers, or multimers with either itself or other proteins, the antigenic efficacy of the invention, including its subsequent use a preventative treatment for disease or disease states, or as an effector for targeting diseased genes. Moreover, the ability to enhance specific characteristics of a protein may also be applicable to changing the characterized activity of an enzyme to an activity completely unrelated to its initially characterized activity. Other desirable enhancements of the invention would be specific to each individual protein, and would thus be well known in the art and contemplated by the present invention.

For example, an engineered serpin may have enhanced serine proteasei inhibitory activity. Alternatively, an engineered serpin may be constitutively active in the absence of substrate binding. In yet another example, an engineered serpin may be capable of being activated with less than all of the regulatory factors and/or conditions typically required for serpin activation (e.g., substrate binding, phosphorylation, conformational changes, etc.). In another example, an engineered serpin may have altered substrate specificity. In yet another example, an engineered serpin may be useful as a suicide inhibitor by covalently binding to its substrate and rendering it permanently inactive. Such serpins would also be useful in screens to identify serpin modulators, among other uses described herein.

Directed evolution is comprised of several steps. The first step is to establish a library of variants for the gene or protein of interest. The most important step is to then select for those variants that entail the activity you wish to identify. The design of the screen is essential since your screen should be selective enough to eliminate non-useful variants, but not so stringent as to eliminate all variants. The last step is then to repeat the above steps using the best variant from the previous screen. Each successive cycle, can then be tailored as necessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed to introduce mutations into macromolecules. Some of these methods include, random mutagenesis, “error-prone” PCR, chemical mutagenesis, site-directed mutagenesis, and other methods well known in the art (for a comprehensive listing of current mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Typically, such methods have been used, for example, as tools for identifying the core functional region(s) of a protein or the function of specific domains of a protein (if a multi-domain protein). However, such methods have more recently been applied to the identification of macromolecule variants with specific or enhanced characteristics.

Random mutagenesis has been the most widely recognized method to date. Typically, this has been carried out either through the use of “error-prone” PCR (as described in Moore, J., et al, Nature Biotechnology 14:458, (1996), or through the application of randomized synthetic oligonucleotides corresponding to specific regions of interest (as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), and Hill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approaches have limits to the level of mutagenesis that can be obtained. However, either approach enables the investigator to effectively control the rate of mutagenesis. This is particularly important considering the fact that mutations beneficial to the activity of the enzyme are fairly rare. In fact, using too high a level of mutagenesis may counter or inhibit the desired benefit of a useful mutation.

While both of the aforementioned methods are effective for creating randomized pools of macromolecule variants, a third method, termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling has also been referred to as “directed molecular evolution”, “exon-shuffling”, “directed enzyme evolution”, “in vitro evolution”, and “artificial evolution”. Such reference terms are known in the art and are encompassed by the invention. This new, preferred, method apparently overcomes the limitations of the previous methods in that it not only propagates positive traits, but simultaneously eliminates negative traits in the resulting progeny.

DNA shuffling accomplishes this task by combining the principal of in vitro recombination, along with the method of “error-prone” PCR. In effect, you begin with a randomly digested pool of small fragments of your gene, created by Dnase I digestion, and then introduce said random fragments into an “error-prone” PCR assembly reaction. During the PCR reaction, the randomly sized DNA fragments not only hybridize to their cognate strand, but also may hybridize to other DNA fragments corresponding to different regions of the polynucleotide of interest—regions not typically accessible via hybridization of the entire polynucleotide. Moreover, since the PCR assembly reaction utilizes “error-prone” PCR reaction conditions, random mutations are introduced during the DNA synthesis step of the PCR reaction for all of the fragments—further diversifying the potential hybridization sites during the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNA shuffling reaction. However, specific reaction conditions for DNA shuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction. Preparation may be in the form of simply purifying the DNA from contaminating cellular material, chemicals, buffers, oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entail the use of DNA purification kits as those provided by Qiagen, Inc., or by the Promega, Corp., for example.

Once the DNA substrate has been purified, it would be subjected to Dnase I digestion. About 2-4 ug of the DNA substrate(s) would be digested with .0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resulting fragments of 10-50 bp could then be purified by running them through a 2% low-melting point agarose gel by electrophoresis onto DE81 ion-exchange paper (Whatmann) or could be purified using Microcon concentrators (Amicon) of the appropriate molecular weight cutoff, or could use oligonucleotide purification columns (Qiagen), in addition to other methods known in the art. If using DE81 ion-exchange paper, the 10-50 bp fragments could be eluted from said paper using 1M NaCl, followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCR assembly reaction by re-suspension in a PCR mixture containing: 2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM TrisoHCL, pH 9.0, and 0.1% Triton X-100, at a final fragment concentration of 10-30 ng/ul. No primers are added at this point. Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 C for 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45 cycles, followed by 72 C for 5 min using an MJ Research (Cambridge, Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a 1:40 dilution of the resulting primerless product would then be introduced into a PCR mixture (using the same buffer mixture used for the assembly reaction) containing 0.8um of each primer and subjecting this mixture to 15 cycles of PCR (using 94 C for 30s, 50 C for 30s, and 72 C for 30s). The referred primers would be primers corresponding to the nucleic acid sequences of the polynucleotide(s) utilized in the shuffling reaction. Said primers could consist of modified nucleic acid base pairs using methods known in the art and referred to else where herein, or could contain additional sequences (i.e., for adding restriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purified using methods well known in the art (e.g., Qiagen PCR purification kits) and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published to date, such variations would be obvious to the skilled artisan and are encompassed by the invention. The DNA shuffling method can also be tailored to the desired level of mutagenesis using the methods described by Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).

As described above, once the randomized pool has been created, it can then be subjected to a specific screen to identify the variant possessing the desired characteristic(s). Once the variant has been identified, DNA corresponding to the variant could then be used as the DNA substrate for initiating another round of DNA shuffling. This cycle of shuffling, selecting the optimized variant of interest, and then re-shuffling, can be repeated until the ultimate variant is obtained. Examples of model screens applied to identify variants created using DNA shuffling technology may be found in the following publications: J. C., Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al., Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat. Biotech., 15:436-438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficial mutations. When combined with screening, DNA shuffling allows the discovery of the best mutational combinations and does not assume that the best combination contains all the mutations in a population. Secondly, recombination occurs simultaneously with point mutagenesis. An effect of forcing DNA polymerase to synthesize full-length genes from the small fragment DNA pool is a background mutagenesis rate. In combination with a stringent selection method, enzymatic activity has been evolved up to 16000 fold increase over the wild-type form of the enzyme. In essence, the background mutagenesis yielded the genetic variability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to remove deleterious mutations. As discussed above, during the process of the randomization, for every one beneficial mutation, there may be at least one or more neutral or inhibitory mutations. Such mutations can be removed by including in the assembly reaction an excess of the wild-type random-size fragments, in addition to the random-size fragments of the selected mutant from the previous selection. During the next selection, some of the most active variants of the polynucleotide/polypeptide/enzyme, should have lost the inhibitory mutations.

Finally, recombination enables parallel processing. This represents a significant advantage since there are likely multiple characteristics that would make a protein more desirable (e.g. solubility, activity, etc.). Since it is increasingly difficult to screen for more than one desirable trait at a time, other methods of molecular evolution tend to be inhibitory. However, using recombination, it would be possible to combine the randomized fragments of the best representative variants for the various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides and polypeptides of the present invention to decrease their immunogenicity in a specified host. For example, a particular variant of the present invention may be created and isolated using DNA shuffling technology. Such a variant may have all of the desired characteristics, though may be highly immunogenic in a host due to its novel intrinsic structure. Specifically, the desired characteristic may cause the polypeptide to have a non-native structure which could no longer be recognized as a “self” molecule, but rather as a “foreign”, and thus activate a host immune response directed against the novel variant. Such a limitation can be overcome, for example, by including a copy of the gene sequence for a xenobiotic ortholog of the native protein in with the gene sequence of the novel variant gene in one or more cycles of DNA shuffling. The molar ratio of the ortholog and novel variant DNAs could be varied accordingly. Ideally, the resulting hybrid variant identified would contain at least some of the coding sequence which enabled the xenobiotic protein to evade the host immune system, and additionally, the coding sequence of the original novel variant that provided the desired characteristics.

Likewise, the invention encompasses the application of DNA shuffling technology to the evolution of polynucleotides and polypeptides of the invention, wherein one or more cycles of DNA shuffling include, in addition to the gene template DNA, oligonucleotides coding for known allelic sequences, optimized codon sequences, known variant sequences, known polynucleotide polymorphism sequences, known ortholog sequences, known homologue sequences, additional homologous sequences, additional non-homologous sequences, sequences from another species, and any number and combination of the above.

In addition to the described methods above, there are a number of related methods that may also be applicable, or desirable in certain cases. Representative among these are the methods discussed in PCT applications WO 98/31700, and WO 98/32845, which are hereby incorporated by reference. Furthermore, related methods can also be applied to the polynucleotide sequences of the present invention in order to evolve invention for creating ideal variants for use in gene therapy, protein engineering, evolution of whole cells containing the variant, or in the evolution of entire enzyme pathways containing polynucleotides of the invention as described in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech., 15:436-438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to the polynucleotides and polypeptides of the present invention, including their proposed applications, may be found in U.S. Pat. No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCT Application No. WO 97/35966; and PCT Application No. WO 98/42832; PCT Application No. WO 00/09727 specifically provides methods for applying DNA shuffling to the identification of herbicide selective crops which could be applied to the polynucleotides and polypeptides of the present invention; additionally, PCT Application No. WO 00/12680 provides methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that confer detectable phenotypic properties on plant species; each of the above are hereby incorporated in their entirety herein for all purposes.

Example 22 Method of Determining Alterations in a Gene Corresponding to a Polynucleotide

RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:1. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.

PCR products is cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C— and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.

Example 23 Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described elsewhere herein. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.

Example 24 Formulation

The invention also provides methods of treatment and/or prevention diseases, disorders, and/or conditions (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant a polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.

Therapeutics can be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention may also be suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see, generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 317-327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.

In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic will typically be formulated in such vehicles at a concentration of about 0.1 mg/mil to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR((zidovudine/AZT), VIDEX((didanosine/ddI), HIVID((zalcitabine/ddC), ZERIT((stavudine/d4T), EPIVIR((lamivudine 3TC), and COMBIVIR( (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE((nevirapine), RESCRIPTOR((delavirdine), and SUSTIVA((efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN( (indinavir), NORVIR((ritonavir), INVIRASE((saquinavir), and VIRACEPT( (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.

In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE(, DAPSONE(, PENTAMIDINE(, ATOVAQUONE(, ISONIAZID(, RIFAMPIN(, PYRAZINAMIDE(, ETHAMBUTOL(, RIFABUTIN(, CLARITHROMYCIN(, AZITHROMYCIN(, GANCICLOVIR(, FOSCARNET(, CIDOFOVIR(, FLUCONAZOLE(, ITRACONAZOLE(, KETOCONAZOLE(, ACYCLOVIR(, FAMCICOLVIR(, PYRIMETHAMINE(, LEUCOVORIN(, NEUPOGEN( (filgrastim/G-CSF), and LEUKINE((sargramostim,GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE(, DAPSONE(, PENTAMIDINE(, and/or ATOVAQUONE( to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID(, RIFAMPIN(, PYRAZINAMIDE(, and/or ETHAMBUTOL( to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN(, CLARITHROMYCIN(, and/or AZITHROMYCIN( to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR(, FOSCARNET(, and/or CIDOFOVIR( to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE(, ITRACONAZOLE(, and/or KETOCONAZOLE( to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR( and/or FAMCICOLVIR( to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE( and/or LEUCOVORIN( to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN( and/or NEUPOGEN( to prophylactically treat or prevent an opportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administered in combination with immunosuppressants. Immunosuppressants preparations that may be administered with the Therapeutics of the invention include, but are not limited to, ORTHOCLONE((OKT3), SANDIMMUNE(/NEORAL(/SANGDYA((cyclosporin), PROGRAF((tacrolimus), CELLCEPT((mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE((sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR(, IVEEGAM(, SANDOGLOBULIN(, GAMMAGARD S/D(, and GAMIMUNE(. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In another embodiment, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or any combination of the components of CHOP. In another embodiment, Therapeutics of the invention are administered in combination with Rituximab. In a further embodiment, Therapeutics of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PlGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, LEUKINE((SARGRAMOSTIM( ) and NEUPOGEN((FILGRASTIM( ).

In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In a specific embodiment, formulations of the present invention may further comprise antagonists of P-glycoprotein (also referred to as the multiresistance protein, or PGP), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). P-glycoprotein is well known for decreasing the efficacy of various drug administrations due to its ability to export intracellular levels of absorbed drug to the cell exterior. While this activity has been particularly pronounced in cancer cells in response to the administration of chemotherapy regimens, a variety of other cell types and the administration of other drug classes have been noted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations in the PGP gene significantly reduces PGP function, making it less able to force drugs out of cells. People who have two versions of the mutated gene—one inherited from each parent—have more than four times less PGP than those with two normal versions of the gene. People may also have one normal gene and one mutated one. Certain ethnic populations have increased incidence of such PGP mutations. Among individuals from Ghana, Kenya, the Sudan, as well as African Americans, frequency of the normal gene ranged from 73% to 84%. In contrast, the frequency was 34% to 59% among British whites, Portuguese, Southwest Asian, Chinese, Filipino and Saudi populations. As a result, certain ethnic populations may require increased administration of PGP antagonist in the formulation of the present invention to arrive at the an efficacious dose of the therapeutic (e.g., those from African descent). Conversely, certain ethnic populations, particularly those having increased frequency of the mutated PGP (e.g., of Caucasian descent, or non-African descent) may require less pharmaceutical compositions in the formulation due to an effective increase in efficacy of such compositions as a result of the increased effective absorption (e.g., less PGP activity) of said composition.

Moreover, in another specific embodiment, formulations of the present invention may further comprise antagonists of OATP2 (also referred to as the multiresistance protein, or MRP2), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). The invention also further comprises any additional antagonists known to inhibit proteins thought to be attributable to a multidrug resistant phenotype in proliferating cells.

In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Example 25 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided herein.

Example 26 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided herein.

Example 27 Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.

At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.

The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 9 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.

The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.

Example 28 Gene Therapy Using Endogenous Genes Corresponding to Polynucleotides of the Invention

Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No: 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation.

In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.

Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×106 cells/ml. Electroporation should be performed immediately following resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1-XbaI; fragment 2-BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×106 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.

The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 29 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.

Example 30 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.

Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 31 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.

In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 32 Production of an Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing LSI-01 are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of LSI-01 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

Monoclonal antibodies specific for protein LSI-01 are prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with LSI-01 polypeptide or, more preferably, with a secreted LSI-01 polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the LSI-01 polypeptide.

Alternatively, additional antibodies capable of binding to LSI-01 polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the LSI-01 protein-specific antibody can be blocked by LSI-01. Such antibodies comprise anti-idiotypic antibodies to the LSI-01 protein-specific antibody and are used to immunize an animal to induce formation of further LSI-01 protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

b) Isolation of Antibody Fragments Directed Against LSI-01 from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against LSI-01 to which the donor may or may not have been exposed (see e.g., U.S. Pat. 5,885,793 incorporated herein by reference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in PCT publication WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see PCT publication WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in PCT publication

M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylarnine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., PCT publication WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.

Example 33 Assays Detecting Stimulation or Inhibition of B cell Proliferation and Differentiation

Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.

In Vitro Assay—Purified polypeptides of the invention, or truncated forms thereof, is assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the polypeptides of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 105 B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10-5M 2ME, 100 U /ml penicillin, 10 ug/ml streptomycin, and 10-5 dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20 h pulse (luCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of a polypeptide of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with polypeptides of the invention identify the results of the activity of the polypeptides on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated with polypeptide is used to indicate whether the polypeptide specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.

Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and polypeptide-treated mice.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 34 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of 3H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 (1/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4 degrees C. (1 (g/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×104/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of polypeptides of the invention (total volume 200 ul). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 degrees C., plates are spun for 2 min. at 1000 rpm and 100 (1 of supernatant is removed and stored −20 degrees C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.5 uCi of 3H-thymidine and cultured at 37 degrees C. for 18-24 hr. Wells are harvested and incorporation of 3H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative controls for the effects of polypeptides of the invention.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 35 Effect of Polypeptides of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-(, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FC(RII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.

FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Th1 helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (106/ml) are treated with increasing concentrations of polypeptides of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit(e.g., R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increase expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Polypeptides, agonists, or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated process (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2×106/ml in PBS containing PI at a final concentration of 5 (g/ml, and then incubated at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.

Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×105 cells/ml with increasing concentrations of the a polypeptide of the invention and under the same conditions, but in the absence of the polypeptide. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in presence of a polypeptide of the invention. LPS (10 ng/ml) is then added. Conditioned media are collected after 24 h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×105 cell/well. Increasing concentrations of polypeptides of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37(C for 2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H202 produced by the macrophages, a standard curve of a H202 solution of known molarity is performed for each experiment.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 36 Biological Effects of Polypeptides of the Invention

Astrocyte and Neuronal Assays.

Recombinant polypeptides of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate a polypeptide of the invention's activity on these cells.

Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of a polypeptide of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.

Fibroblast and Endothelial Cell Assays.

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE2 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or polypeptides of the invention with or without IL-1( for 24 hours. The supernatants are collected and assayed for PGE2 by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without polypeptides of the invention IL-1( for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which can be used to compare stimulation with polypeptides of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released. Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex 1), thereby interfering with electron transport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of a polypeptide of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 37 The Effect of Polypeptides of the Invention on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5×104 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. A polypeptide having the amino acid sequence of SEQ ID NO:2, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the polypeptide of the invention may proliferate vascular endothelial cells.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 38 Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed to incubate for 1 hour at 37° C. before measuring the absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) is subtracted, and seven wells are performed in parallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994).

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 39 Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd Staining Kit (Zymed Laboratories). In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposed to denaturing solution and then incubated with the streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a percent of the BrdUrd-positive cells to the total cell number. In addition, the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271(36):21985-21992 (1996).

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 40 Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W., et al., J. Immunological Methods 1980;33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5×105 cells suspended in 50 ul M199 containing 1% FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37° C. in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration is quantified by counting cells of three random high-power fields (40×) in each well, and all groups are performed in quadruplicate.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 41 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive control (such as VEGF-1) and the polypeptide of the invention. Nitric oxide in the medium is determined by use of the Griess reagent to measure total nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of the polypeptide of the invention on nitric oxide release is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with a NO-specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NO elements is performed according to the following equation: 2KNO2+2KI+2H2SO4 6 2NO+12+2H2O+2K2SO4

The standard calibration curve is obtained by adding graded concentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration solution containing KI and H2SO4. The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050). The culture medium is removed and HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells are then bathed in 5 mil of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates are kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature at 37° C. The NO sensor probe is inserted vertically into the wells, keeping the tip of the electrode 2 mm under the surface of the solution, before addition of the different conditions. S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount of released NO is expressed as picomoles per 1×106 endothelial cells. All values reported are means of four to six measurements in each group (number of cell culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 42 Effect of Polypepides of the Invention on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked by differentiation of endothelial cells. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from Cell Applications, Inc. as proliferating (passage 2) cells and are cultured in Cell Applications' CADMEC Growth Medium and used at passage 5. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate are coated with Cell Applications' Attachment Factor Medium (200 ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wells at 7,500 cells/well and cultured overnight in Growth Medium. The Growth Medium is then replaced with 300 mg Cell Applications' Chord Formation Medium containing control buffer or a polypeptide of the invention (0.1 to 100 ng/ml) and the cells are cultured for an additional 48 hr. The numbers and lengths of the capillary-like chords are quantitated through use of the Boeckeler VIA-170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control. b-esteradiol (1 ng/ml) is used as a negative control. The appropriate buffer (without protein) is also utilized as a control.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 43 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system to examine angiogenesis. Blood vessel formation on CAM is easily visible and quantifiable. The ability of polypeptides of the invention to stimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and the Japanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old qual embryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chick eggs. The embryos are checked for normal development and the eggs sealed with cellotape. They are further incubated until Day 13. Thermanox coverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm in diameter. Sterile and salt-free growth factors are dissolved in distilled water and about 3.3 mg/5 ml are pipetted on the disks. After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. They are photographed with a stereo microscope [Wild M8] and embedded for semi- and ultrathin sectioning as described above. Controls are performed with carrier disks alone.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 44 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of a polypeptide of the invention measures the ability of an existing capillary network to form new vessels in an implanted capsule of murine extracellular matrix material (Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C. and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid “plug” of Matrigel is removed and examined for the presence of new blood vessels. Matrigel is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. The Matrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C. and drawn into cold 3 ml syringes. Female C57B1/6 mice approximately 8 weeks old are injected with the mixture of Matrigel and experimental protein at 2 sites at the midventral aspect of the abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical dislocation, the Matrigel plugs are removed and cleaned (i.e., all clinging membranes and fibrous tissue is removed). Replicate whole plugs are fixed in neutral buffered 10% formaldehyde, embedded in paraffin and used to produce sections for histological examination after staining with Masson's Trichrome. Cross sections from 3 different regions of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay is bovine basic FGF (150 ng/ml). Matrigel alone is used to determine basal levels of angiogenesis.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 45 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model is created by surgical removal of one femoral arteries as described previously (Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshitaet al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. At 10 day post-operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90:936-944 (1992)). When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 min. through an infusion catheter. On day 30, various parameters are measured in these rabbits: (a) BP ratio—The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flow during undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL: resting FL; (c) Angiographic Score—This is measured by the angiogram of collateral vessels. A score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh; (d) Capillary density—The number of collateral capillaries determined in light microscopic sections taken from hindlimbs.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 46 Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing blood pressure, the ability of polypeptides of the invention to affect the blood pressure in spontaneously hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as the mean±SEM. Statistical analysis are performed with a paired t-test and statistical significance is defined as p<0.05 vs. the response to buffer alone.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 47 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybridization.

The study in this model is divided into three parts as follows:

a) Ischemic skin

b) Ischemic skin wounds

c) Normal wounds

The experimental protocol includes:

a) Raising a 3×4 cm, single pedicle full-thickness random skin flap (myocutaneous flap over the lower back of the animal).

b) An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-flap).

c) Topical treatment with a polypeptide of the invention of the excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges: 1 mg to 100 mg.

d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-wounding for histological, immunohistochemical, and in situ studies.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 48 Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases. The experimental protocol includes:

a) One side of the femoral artery is ligated to create ischemic muscle of the hindlimb, the other side of hindlimb serves as a control.

b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks.

c) The ischemic muscle tissue is collected after ligation of the femoral artery at 1, 2, and 3 weeks for the analysis of expression of a polypeptide of the invention and histology. Biopsy is also performed on the other side of normal muscle of the contralateral hindlimb.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 49 Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion. Alteration of expression of the polypeptide is investigated in situ. The experimental protocol includes:

a) The heart is exposed through a left-side thoracotomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed.

b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4 weeks.

c) Thirty days after the surgery, the heart is removed and cross-sectioned for morphometric and in situ analyzes.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 50 Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention on neovascularization. The experimental protocol includes:

a) Making a 1-1.5 mm long incision from the center of cornea into the stromal layer.

b) Inserting a spatula below the lip of the incision facing the outer corner of the eye.

c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).

d) Positioning a pellet, containing 50 ng-5 ug of a polypeptide of the invention, within the pocket.

e) Treatment with a polypeptide of the invention can also be applied topically to the corneal wounds in a dosage range of 20 mg-500 mg (daily treatment for five days).

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 51 Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models

A. Diabetic db+/db+ Mouse Model.

To demonstrate that a polypeptide of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Bristol-Myers Squibb Company's Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

A polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula: [Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with a polypeptide of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer can serve as a positive tissue control and human brain tissue can be used as a negative tissue control. Each specimen includes a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahlet al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).

To demonstrate that a polypeptide of the invention can accelerate the healing process, the effects of multiple topical applications of the polypeptide on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study would be conducted according to the rules and guidelines of Bristol-Myers Squibb Corporations Guidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

The polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula: [Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with a polypeptide of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 52 Lymphedema Animal Model

The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of a polypeptide of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ˜350 g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan'Blue. Circumference and volumetric measurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then and ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.

Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (A J Buck). The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of ˜0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.

To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people then those 2 readings are averaged. Readings are taken from both control and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into instrument to each marked level then measured by Buxco edema software(Chen/Victor). Data is recorded by one person, while the other is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at −80EC until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 53 Suppression of TNF Alpha-Induced Adhesion Molecule Expression by a Polypeptide of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-I (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.

The potential of a polypeptide of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a member of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidified incubator containing 5% CO2. HUVECs are seeded in 96-well plates at concentrations of 1×104 cells/well in EGM medium at 37 degree C. for 18-24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 ul volumes). Plates are incubated at 37 degree C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 μl of diluted primary antibody to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphatase (1:5,000 dilution) to each well and incubated at 37° C. for 30 min. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphatase in glycine buffer: 1:5,000 (100)>10−0.5>10−1>10−1.5. 5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 54 Method of Chromosomal Mapping of Serpin LS-1 in 14q32.1 Among a Cluster of Proteases in a Region Prone to Rearrangement Causing Leukemias

A chromosomal localization of the Serpin LS-1 gene was performed by using the nucleic acid sequence of the invention (SEQ ID NO:1) in a database search of the recently completed draft of human genome sequence. Using the Basic Local Alignment Search Tool 2 (BLAST2), the first 200 bp of Serpin LS-1 cDNA sequence (SEQ ID NO:1) showed a perfect alignment of nucleotides 12 to 104 and 103 to 200 with Homo sapiens chromosome 14 Bac clones R-262P9 (gi|AL132990) and R-34911 (gi|AL132708), suggesting the presence of two introns. Despite the absence of map data for Bac R-262P9, Bac R-34911 was already registered in the clone database (NCBI), and mapped by the Genoscope center (France), to 14q32. 1. AL132708 is part of NCBI contig NT_(—)019573 that already contains 6 serpins (A1-A6) and a protease Z inhibitor. This chromosomal region is commonly involved in chromosomal rearrangements, inversion, or translocations associated with the incidence of T-cell malignancy (T-cell neoplasms, leukemias; Saitou et al. Identification of the TCL6 genes within the breakpoint cluster region on chromosome 14q32 in T-cell leukemia. Oncogene 19: 2796-2802, 2000.), or B-cell leukemias (i.e. one of the most frequent hematologic malignancies, follicular lymphoma is associated with a reciprocal translocation t(14;18)(q32;q21) and can result in deregulated expression of a chromosome 18 gene, Bc12, which interferes with normal apoptosis of B lymphocytes; Ngan, B.-Y. et al. Expression in non-Hodgkin's lymphoma of the BCL-2 protein associated with the t(14;18) chromosomal translocation. New Eng. J. Med. 318:1638-1644, 1988). The oncogene activated by these different rearrangements is not always the same, and is dependent upon where the chromosomal breakpoints occur. Several candidate oncogenes responsible for malignant transformation have already been identified in chromosome 14q32 (i.e. TCL1A (T-cell leukemia/lymphoma 1a), TCL1b, TCL6, TRIP11(Thyroid Hormone Receptor Interactor 11)).

Based on its expression profile and genetic map, Serpin LS-1 could be a novel specific biomarker that could pinpoint a subgroup of patients with chronic lymphocytic leukemias (with a distinct prognostic).

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties. TABLE III Atom Atom Type Residue # X Y Z B Occ ATOM 1 N MET A 1 15.398 13.285 −2.442 1.00 51.19 ATOM 5 CA MET A 1 14.789 12.112 −3.100 1.00 50.26 ATOM 6 CB MET A 1 13.488 12.499 −3.787 1.00 48.58 ATOM 7 CG MET A 1 12.513 13.187 −2.840 1.00 48.82 ATOM 8 SD MET A 1 10.963 13.735 −3.590 1.00 48.56 ATOM 9 CE MET A 1 10.258 14.614 −2.177 1.00 48.52 ATOM 10 C MET A 1 15.740 11.549 −4.150 1.00 49.68 ATOM 11 O MET A 1 16.289 12.312 −4.954 1.00 49.45 ATOM 12 N GLN A 2 15.955 10.244 −4.112 1.00 49.48 ATOM 14 CA GLN A 2 16.809 9.589 −5.115 1.00 48.97 ATOM 15 CB GLN A 2 18.283 9.898 −4.815 1.00 48.89 ATOM 16 CG GLN A 2 19.293 9.196 −5.738 1.00 48.48 ATOM 17 CD GLN A 2 19.133 9.555 −7.225 1.00 47.98 ATOM 18 OE1 GLN A 2 18.028 9.792 −7.725 1.00 47.57 ATOM 19 NE2 GLN A 2 20.227 9.435 −7.952 1.00 48.08 ATOM 22 C GLN A 2 16.567 8.076 −5.192 1.00 48.31 ATOM 23 O GLN A 2 16.528 7.372 −4.174 1.00 48.16 ATOM 24 N GLY A 3 16.442 7.592 −6.417 1.00 48.01 ATOM 26 CA GLY A 3 16.245 6.173 −6.683 1.00 47.41 ATOM 27 C GLY A 3 17.482 5.483 −7.254 1.00 46.87 ATOM 28 O GLY A 3 18.162 5.975 −8.166 1.00 46.65 ATOM 29 N GLN A 4 17.791 4.349 −6.657 1.00 46.75 ATOM 31 CA GLN A 4 18.825 3.454 −7.179 1.00 46.31 ATOM 32 CB GLN A 4 19.438 2.741 −5.984 1.00 46.49 ATOM 33 CG GLN A 4 20.569 1.810 −6.385 1.00 47.05 ATOM 34 CD GLN A 4 20.529 0.605 −5.459 1.00 47.56 ATOM 35 OE1 GLN A 4 21.435 0.388 −4.647 1.00 48.03 ATOM 36 NE2 GLN A 4 19.465 −0.166 −5.603 1.00 47.50 ATOM 39 C GLN A 4 18.192 2.419 −8.114 1.00 45.37 ATOM 40 O GLN A 4 17.816 1.319 −7.683 1.00 45.03 ATOM 41 N GLY A 5 18.153 2.742 −9.395 1.00 45.01 ATOM 43 CA GLY A 5 17.485 1.863 −10.363 1.00 44.13 ATOM 44 C GLY A 5 17.931 2.078 −11.807 1.00 43.80 ATOM 45 O GLY A 5 17.182 2.627 −12.630 1.00 43.85 ATOM 46 N ARG A 6 18.987 1.366 −12.159 1.00 44.78 ATOM 48 CA ARG A 6 19.560 1.419 −13.511 1.00 44.72 ATOM 49 CB ARG A 6 20.840 0.577 −13.455 1.00 45.73 ATOM 50 CG ARG A 6 21.653 0.544 −14.748 1.00 46.28 ATOM 51 CD ARG A 6 21.214 −0.551 −15.717 1.00 46.92 ATOM 52 NE ARG A 6 21.341 −1.878 −15.096 1.00 47.35 ATOM 53 CZ ARG A 6 21.802 −2.945 −15.751 1.00 47.43 ATOM 54 NH1 ARG A 6 22.185 −2.829 −17.025 1.00 47.60 ATOM 55 NH2 ARG A 6 21.883 −4.125 −15.132 1.00 47.39 ATOM 56 C ARG A 6 18.570 0.859 −14.532 1.00 43.76 ATOM 57 O ARG A 6 18.222 1.569 −15.490 1.00 43.44 ATOM 58 N ARG A 7 17.854 −0.164 −14.079 1.00 43.34 ATOM 60 CA ARG A 7 16.899 −0.917 −14.899 1.00 42.46 ATOM 61 CB ARG A 7 16.641 −2.243 −14.194 1.00 43.40 ATOM 62 CG ARG A 7 17.924 −2.971 −13.819 1.00 43.50 ATOM 63 CD ARG A 7 17.598 −4.239 −13.038 1.00 43.72 ATOM 64 NE ARG A 7 18.816 −4.938 −12.600 1.00 43.80 ATOM 65 CZ ARG A 7 19.090 −5.179 −11.316 1.00 43.65 ATOM 66 NH1 ARG A 7 18.302 −4.680 −10.360 1.00 43.77 ATOM 67 NH2 ARG A 7 20.196 −5.849 −10.985 1.00 43.45 ATOM 68 C ARG A 7 15.539 −0.239 −15.071 1.00 41.88 ATOM 69 O ARG A 7 14.728 −0.728 −15.865 1.00 42.14 ATOM 70 N ARG A 8 15.292 0.874 −14.395 1.00 41.14 ATOM 72 CA ARG A 8 13.995 1.533 −14.557 1.00 40.60 ATOM 73 CB ARG A 8 13.541 2.104 −13.214 1.00 40.61 ATOM 74 CG ARG A 8 14.151 3.454 −12.848 1.00 40.15 ATOM 75 CD ARG A 8 13.879 3.761 −11.379 1.00 39.95 ATOM 76 NE ARG A 8 13.957 5.197 −11.056 1.00 40.56 ATOM 77 CZ ARG A 8 15.044 5.843 −10.627 1.00 41.08 ATOM 78 NH1 ARG A 8 14.906 6.999 −9.975 1.00 41.39 ATOM 79 NH2 ARG A 8 16.212 5.210 −10.572 1.00 41.34 ATOM 80 C ARG A 8 14.071 2.601 −15.647 1.00 39.99 ATOM 81 O ARG A 8 13.045 3.140 −16.077 1.00 39.81 ATOM 82 N GLY A 9 15.272 2.814 −16.163 1.00 39.73 ATOM 84 CA GLY A 9 15.471 3.775 −17.243 1.00 39.19 ATOM 85 C GLY A 9 16.382 4.900 −16.789 1.00 38.79 ATOM 86 O GLY A 9 16.113 6.082 −17.041 1.00 38.36 ATOM 87 N THR A 10 17.431 4.530 −16.078 1.00 39.03 ATOM 89 CA THR A 10 18.377 5.532 −15.590 1.00 38.73 ATOM 90 CB THR A 10 18.584 5.253 −14.107 1.00 39.28 ATOM 91 OG1 THR A 10 17.312 5.377 −13.488 1.00 39.82 ATOM 92 CG2 THR A 10 19.536 6.238 −13.440 1.00 39.49 ATOM 93 C THR A 10 19.699 5.477 −16.353 1.00 38.15 ATOM 94 O THR A 10 20.441 6.463 −16.406 1.00 38.33 ATOM 95 N CYS A 11 19.936 4.357 −17.016 1.00 37.53 ATOM 97 CA CYS A 11 21.187 4.185 −17.763 1.00 36.98 ATOM 98 CB CYS A 11 21.704 2.781 −17.468 1.00 37.21 ATOM 99 SG CYS A 11 23.381 2.392 −18.024 1.00 37.18 ATOM 100 C CYS A 11 21.015 4.373 −19.274 1.00 36.77 ATOM 101 O CYS A 11 21.989 4.257 −20.025 1.00 36.52 ATOM 102 N LYS A 12 19.804 4.660 −19.722 1.00 37.20 ATOM 104 CA LYS A 12 19.569 4.726 −21.169 1.00 37.21 ATOM 105 CB LYS A 12 18.372 3.848 −21.522 1.00 37.42 ATOM 106 CG LYS A 12 17.097 4.333 −20.845 1.00 37.48 ATOM 107 CD LYS A 12 15.883 3.547 −21.326 1.00 38.13 ATOM 108 CE LYS A 12 14.599 4.079 −20.702 1.00 38.28 ATOM 109 NZ LYS A 12 13.424 3.339 −21.186 1.00 38.36 ATOM 110 C LYS A 12 19.323 6.148 −21.670 1.00 36.94 ATOM 111 O LYS A 12 18.893 7.025 −20.918 1.00 36.54 ATOM 112 N ASP A 13 19.566 6.316 −22.963 1.00 37.27 ATOM 114 CA ASP A 13 19.263 7.542 −23.737 1.00 37.15 ATOM 115 CB ASP A 13 17.796 7.492 −24.158 1.00 37.99 ATOM 116 CG ASP A 13 17.537 6.285 −25.053 1.00 38.69 ATOM 117 OD1 ASP A 13 17.088 5.272 −24.533 1.00 39.33 ATOM 118 OD2 ASP A 13 17.786 6.402 −26.245 1.00 38.66 ATOM 119 C ASP A 13 19.536 8.871 −23.028 1.00 36.89 ATOM 120 O ASP A 13 20.506 8.992 −22.272 1.00 37.25 ATOM 121 N ILE A 14 18.749 9.863 −23.436 1.00 36.29 ATOM 123 CA ILE A 14 18.784 11.283 −23.004 1.00 36.08 ATOM 124 CB ILE A 14 17.594 11.554 −22.087 1.00 36.68 ATOM 125 CG2 ILE A 14 17.672 12.935 −21.442 1.00 36.42 ATOM 126 CG1 ILE A 14 16.300 11.424 −22.883 1.00 37.41 ATOM 127 CD1 ILE A 14 15.092 11.878 −22.071 1.00 38.01 ATOM 128 C ILE A 14 20.089 11.781 −22.384 1.00 35.41 ATOM 129 O ILE A 14 20.477 11.412 −21.270 1.00 35.11 ATOM 130 N PHE A 15 20.678 12.723 −23.108 1.00 35.24 ATOM 132 CA PHE A 15 21.982 13.323 −22.792 1.00 34.77 ATOM 133 CB PHE A 15 22.080 14.603 −23.618 1.00 35.12 ATOM 134 CG PHE A 15 23.432 15.309 −23.595 1.00 35.04 ATOM 135 CD1 PHE A 15 24.604 14.583 −23.764 1.00 35.07 ATOM 136 CE1 PHE A 15 25.832 15.232 −23.748 1.00 35.04 ATOM 137 CZ PHE A 15 25.887 16.608 −23.570 1.00 35.02 ATOM 138 CE2 PHE A 15 24.715 17.336 −23.410 1.00 34.98 ATOM 139 CD2 PHE A 15 23.488 16.686 −23.426 1.00 34.98 ATOM 140 C PHE A 15 22.145 13.648 −21.308 1.00 34.03 ATOM 141 O PHE A 15 21.313 14.340 −20.704 1.00 33.59 ATOM 142 N CYS A 16 23.133 12.977 −20.730 1.00 33.95 ATOM 144 CA CYS A 16 23.581 13.118 −19.330 1.00 33.34 ATOM 145 CB CYS A 16 23.688 14.595 −18.951 1.00 33.26 ATOM 146 SG CYS A 16 24.914 15.560 −19.865 1.00 33.25 ATOM 147 C CYS A 16 22.733 12.386 −18.278 1.00 32.74 ATOM 148 O CYS A 16 23.314 11.677 −17.447 1.00 32.44 ATOM 149 N SER A 17 21.415 12.532 −18.300 1.00 32.60 ATOM 151 CA SER A 17 20.591 11.978 −17.211 1.00 32.03 ATOM 152 CB SER A 17 20.891 12.787 −15.950 1.00 32.30 ATOM 153 OG SER A 17 21.022 14.156 −16.322 1.00 32.53 ATOM 154 C SER A 17 19.084 11.984 −17.487 1.00 32.04 ATOM 155 O SER A 17 18.593 11.378 −18.447 1.00 32.34 ATOM 156 N LYS A 18 18.369 12.606 −16.558 1.00 33.24 ATOM 158 CA LYS A 18 16.905 12.756 −16.602 1.00 32.73 ATOM 159 CB LYS A 18 16.501 13.572 −17.824 1.00 33.18 ATOM 160 CG LYS A 18 17.080 14.978 −17.749 1.00 33.24 ATOM 161 CD LYS A 18 16.657 15.820 −18.945 1.00 33.87 ATOM 162 CE LYS A 18 17.230 17.230 −18.853 1.00 34.03 ATOM 163 NZ LYS A 18 16.800 18.049 −19.998 1.00 34.59 ATOM 164 C LYS A 18 16.170 11.422 −16.584 1.00 32.46 ATOM 165 O LYS A 18 16.295 10.659 −15.619 1.00 32.34 ATOM 166 N MET A 19 15.468 11.137 −17.670 1.00 32.43 ATOM 168 CA MET A 19 14.544 9.991 −17.789 1.00 32.25 ATOM 169 CB MET A 19 15.269 8.820 −18.433 1.00 33.06 ATOM 170 CG MET A 19 15.850 9.218 −19.778 1.00 33.76 ATOM 171 SD MET A 19 15.922 7.897 −21.003 1.00 34.22 ATOM 172 CE MET A 19 14.148 7.643 −21.234 1.00 34.73 ATOM 173 C MET A 19 13.934 9.527 −16.469 1.00 32.00 ATOM 174 O MET A 19 13.467 10.345 −15.661 1.00 32.06 ATOM 175 N ALA A 20 14.101 8.249 −16.170 1.00 31.80 ATOM 177 CA ALA A 20 13.431 7.674 −14.999 1.00 31.62 ATOM 178 CB ALA A 20 13.386 6.164 −15.140 1.00 31.45 ATOM 179 C ALA A 20 14.059 8.048 −13.658 1.00 30.71 ATOM 180 O ALA A 20 13.333 8.045 −12.657 1.00 30.35 ATOM 181 N SER A 21 15.235 8.662 −13.683 1.00 30.45 ATOM 183 CA SER A 21 15.855 9.132 −12.442 1.00 29.65 ATOM 184 CB SER A 21 17.371 9.206 −12.557 1.00 29.99 ATOM 185 OG SER A 21 17.709 10.386 −13.291 1.00 29.93 ATOM 186 C SER A 21 15.307 10.501 −12.037 1.00 29.12 ATOM 187 O SER A 21 15.765 11.067 −11.043 1.00 28.77 ATOM 188 N TYR A 22 14.426 11.070 −12.845 1.00 29.15 ATOM 190 CA TYR A 22 13.652 12.235 −12.410 1.00 28.75 ATOM 191 CB TYR A 22 13.788 13.365 −13.429 1.00 28.70 ATOM 192 CG TYR A 22 15.018 14.267 −13.299 1.00 28.83 ATOM 193 CD1 TYR A 22 16.292 13.734 −13.147 1.00 29.48 ATOM 194 CE1 TYR A 22 17.391 14.574 −13.034 1.00 29.69 ATOM 195 CZ TYR A 22 17.213 15.950 −13.077 1.00 29.26 ATOM 196 OH TYR A 22 18.302 16.786 −12.968 1.00 29.57 ATOM 197 CE2 TYR A 22 15.943 16.489 −13.231 1.00 28.61 ATOM 198 CD2 TYR A 22 14.845 15.645 −13.344 1.00 28.39 ATOM 199 C TYR A 22 12.181 11.853 −12.280 1.00 28.19 ATOM 200 O TYR A 22 11.509 12.233 −11.307 1.00 27.76 ATOM 201 N LEU A 23 11.777 10.910 −13.120 1.00 28.27 ATOM 203 CA LEU A 23 10.363 10.512 −13.228 1.00 27.85 ATOM 204 CB LEU A 23 10.153 9.796 −14.558 1.00 28.59 ATOM 205 CG LEU A 23 10.320 10.731 −15.750 1.00 28.61 ATOM 206 CD1 LEU A 23 10.253 9.952 −17.061 1.00 29.14 ATOM 207 CD2 LEU A 23 9.276 11.843 −15.732 1.00 28.27 ATOM 208 C LEU A 23 9.855 9.602 −12.111 1.00 27.38 ATOM 209 O LEU A 23 8.636 9.458 −11.966 1.00 26.72 ATOM 210 N TYR A 24 10.740 9.103 −11.263 1.00 27.81 ATOM 212 CA TYR A 24 10.310 8.300 −10.111 1.00 27.64 ATOM 213 CB TYR A 24 11.392 7.268 −9.828 1.00 28.21 ATOM 214 CG TYR A 24 10.974 6.095 −8.948 1.00 28.08 ATOM 215 CD1 TYR A 24 11.905 5.494 −8.113 1.00 28.38 ATOM 216 CE1 TYR A 24 11.531 4.421 −7.316 1.00 28.36 ATOM 217 CZ TYR A 24 10.227 3.951 −7.356 1.00 28.03 ATOM 218 OH TYR A 24 9.845 2.938 −6.504 1.00 28.10 ATOM 219 CE2 TYR A 24 9.296 4.543 −8.197 1.00 27.70 ATOM 220 CD2 TYR A 24 9.672 5.614 −8.996 1.00 27.73 ATOM 221 C TYR A 24 10.041 9.162 −8.865 1.00 27.22 ATOM 222 O TYR A 24 9.989 8.641 −7.745 1.00 27.04 ATOM 223 N GLY A 25 9.915 10.467 −9.049 1.00 27.18 ATOM 225 CA GLY A 25 9.673 11.366 −7.922 1.00 26.90 ATOM 226 C GLY A 25 11.001 11.758 −7.294 1.00 26.20 ATOM 227 O GLY A 25 11.223 11.567 −6.092 1.00 25.81 ATOM 228 N VAL A 26 11.902 12.243 −8.129 1.00 26.15 ATOM 230 CA VAL A 26 13.225 12.643 −7.641 1.00 25.61 ATOM 231 CB VAL A 26 14.291 11.883 −8.423 1.00 26.41 ATOM 232 CG1 VAL A 26 15.697 12.336 −8.049 1.00 26.69 ATOM 233 CG2 VAL A 26 14.147 10.379 −8.231 1.00 26.42 ATOM 234 C VAL A 26 13.416 14.148 −7.797 1.00 25.10 ATOM 235 O VAL A 26 13.750 14.631 −8.886 1.00 24.61 ATOM 236 N LEU A 27 13.141 14.875 −6.724 1.00 25.30 ATOM 238 CA LEU A 27 13.296 16.339 −6.721 1.00 24.97 ATOM 239 CB LEU A 27 12.039 17.034 −7.237 1.00 25.66 ATOM 240 CG LEU A 27 11.990 17.123 −8.758 1.00 26.01 ATOM 241 CD1 LEU A 27 10.728 17.838 −9.219 1.00 26.79 ATOM 242 CD2 LEU A 27 13.229 17.824 −9.307 1.00 25.84 ATOM 243 C LEU A 27 13.601 16.894 −5.334 1.00 24.45 ATOM 244 O LEU A 27 13.017 16.487 −4.323 1.00 24.15 ATOM 245 N PHE A 28 14.463 17.895 −5.323 1.00 24.45 ATOM 247 CA PHE A 28 14.796 18.618 −4.091 1.00 24.08 ATOM 248 CB PHE A 28 16.252 19.078 −4.203 1.00 24.22 ATOM 249 CG PHE A 28 16.754 20.007 −3.096 1.00 24.07 ATOM 250 CD1 PHE A 28 16.812 19.571 −1.779 1.00 24.31 ATOM 251 CE1 PHE A 28 17.271 20.426 −0.786 1.00 24.27 ATOM 252 CZ PHE A 28 17.677 21.714 −1.110 1.00 24.01 ATOM 253 CE2 PHE A 28 17.630 22.147 −2.427 1.00 23.74 ATOM 254 CD2 PHE A 28 17.172 21.292 −3.420 1.00 23.76 ATOM 255 C PHE A 28 13.853 19.809 −3.923 1.00 23.38 ATOM 256 O PHE A 28 14.109 20.890 −4.466 1.00 22.93 ATOM 257 N ALA A 29 12.739 19.577 −3.244 1.00 23.41 ATOM 259 CA ALA A 29 11.760 20.650 −3.022 1.00 22.89 ATOM 260 CB ALA A 29 11.077 20.995 −4.343 1.00 23.75 ATOM 261 C ALA A 29 10.686 20.270 −2.009 1.00 22.80 ATOM 262 O ALA A 29 10.338 19.095 −1.852 1.00 22.68 ATOM 263 N VAL A 30 10.163 21.304 −1.364 1.00 23.63 ATOM 265 CA VAL A 30 9.004 21.219 −0.450 1.00 23.70 ATOM 266 CB VAL A 30 7.806 20.619 −1.189 1.00 23.97 ATOM 267 CG1 VAL A 30 6.649 20.312 −0.241 1.00 24.62 ATOM 268 CG2 VAL A 30 7.343 21.542 −2.313 1.00 23.77 ATOM 269 C VAL A 30 9.279 20.458 0.849 1.00 23.16 ATOM 270 O VAL A 30 9.463 19.235 0.863 1.00 23.04 ATOM 271 N GLY A 31 9.271 21.215 1.936 1.00 22.80 ATOM 273 CA GLY A 31 9.406 20.648 3.280 1.00 22.28 ATOM 274 C GLY A 31 8.442 21.328 4.252 1.00 21.88 ATOM 275 O GLY A 31 7.225 21.116 4.214 1.00 21.64 ATOM 276 N LEU A 32 9.007 22.129 5.138 1.00 21.78 ATOM 278 CA LEU A 32 8.207 22.877 6.117 1.00 21.43 ATOM 279 CB LEU A 32 8.544 22.349 7.515 1.00 22.42 ATOM 280 CG LEU A 32 7.599 22.865 8.597 1.00 22.76 ATOM 281 CD1 LEU A 32 6.170 22.410 8.334 1.00 23.25 ATOM 282 CD2 LEU A 32 8.060 22.407 9.976 1.00 22.89 ATOM 283 C LEU A 32 8.547 24.365 6.014 1.00 20.99 ATOM 284 O LEU A 32 7.801 25.152 5.414 1.00 20.51 ATOM 285 N CYS A 33 9.712 24.716 6.533 1.00 21.11 ATOM 287 CA CYS A 33 10.181 26.103 6.497 1.00 20.83 ATOM 288 CB CYS A 33 10.731 26.453 7.873 1.00 21.69 ATOM 289 SG CYS A 33 9.540 26.401 9.225 1.00 21.95 ATOM 290 C CYS A 33 11.280 26.263 5.454 1.00 20.22 ATOM 291 O CYS A 33 12.394 25.764 5.641 1.00 20.04 ATOM 292 N ALA A 34 10.950 26.901 4.340 1.00 20.02 ATOM 294 CA ALA A 34 11.963 27.127 3.294 1.00 19.55 ATOM 295 CB ALA A 34 12.020 25.927 2.347 1.00 20.28 ATOM 296 C ALA A 34 11.870 28.478 2.542 1.00 19.00 ATOM 297 O ALA A 34 12.598 29.398 2.938 1.00 18.64 ATOM 298 N PRO A 35 10.921 28.691 1.630 1.00 19.10 ATOM 299 CA PRO A 35 11.203 29.563 0.471 1.00 18.67 ATOM 300 CB PRO A 35 9.981 29.495 −0.396 1.00 18.52 ATOM 301 CG PRO A 35 8.995 28.499 0.185 1.00 18.81 ATOM 302 CD PRO A 35 9.668 27.947 1.430 1.00 19.17 ATOM 303 C PRO A 35 11.525 31.010 0.840 1.00 18.54 ATOM 304 O PRO A 35 12.696 31.356 1.047 1.00 18.21 ATOM 305 N ILE A 36 10.488 31.759 1.181 1.00 19.59 ATOM 307 CA ILE A 36 10.641 33.187 1.498 1.00 19.45 ATOM 308 CB ILE A 36 9.262 33.831 1.382 1.00 19.67 ATOM 309 CG2 ILE A 36 9.274 35.299 1.805 1.00 19.81 ATOM 310 CG1 ILE A 36 8.739 33.712 −0.043 1.00 20.01 ATOM 311 CD1 ILE A 36 9.622 34.488 −1.016 1.00 20.78 ATOM 312 C ILE A 36 11.232 33.429 2.891 1.00 19.16 ATOM 313 O ILE A 36 11.830 34.483 3.116 1.00 19.14 ATOM 314 N TYR A 37 11.370 32.359 3.656 1.00 19.07 ATOM 316 CA TYR A 37 11.889 32.423 5.020 1.00 18.90 ATOM 317 CB TYR A 37 11.235 31.245 5.735 1.00 19.62 ATOM 318 CG TYR A 37 11.389 31.176 7.249 1.00 20.13 ATOM 319 CD1 TYR A 37 11.392 32.330 8.019 1.00 20.53 ATOM 320 CE1 TYR A 37 11.519 32.240 9.398 1.00 21.08 ATOM 321 CZ TYR A 37 11.632 30.996 10.000 1.00 21.23 ATOM 322 OH TYR A 37 11.783 30.899 11.364 1.00 21.89 ATOM 323 CE2 TYR A 37 11.615 29.843 9.233 1.00 20.85 ATOM 324 CD2 TYR A 37 11.488 29.934 7.857 1.00 20.30 ATOM 325 C TYR A 37 13.418 32.291 5.031 1.00 18.56 ATOM 326 O TYR A 37 14.058 32.505 6.068 1.00 18.70 ATOM 327 N CYS A 38 13.973 31.887 3.897 1.00 18.26 ATOM 329 CA CYS A 38 15.424 31.862 3.711 1.00 18.03 ATOM 330 CB CYS A 38 15.805 30.492 3.156 1.00 18.35 ATOM 331 SG CYS A 38 17.572 30.172 2.943 1.00 19.04 ATOM 332 C CYS A 38 15.860 32.949 2.731 1.00 17.59 ATOM 333 O CYS A 38 17.037 33.330 2.693 1.00 17.59 ATOM 334 N VAL A 39 14.908 33.454 1.960 1.00 17.35 ATOM 336 CA VAL A 39 15.210 34.551 1.035 1.00 17.03 ATOM 337 CB VAL A 39 14.194 34.530 −0.105 1.00 17.39 ATOM 338 CG1 VAL A 39 14.333 35.745 −1.018 1.00 17.82 ATOM 339 CG2 VAL A 39 14.314 33.244 −0.913 1.00 17.41 ATOM 340 C VAL A 39 15.149 35.880 1.781 1.00 16.57 ATOM 341 O VAL A 39 15.960 36.786 1.549 1.00 16.13 ATOM 342 N SER A 40 14.231 35.949 2.726 1.00 16.82 ATOM 344 CA SER A 40 14.179 37.070 3.654 1.00 16.58 ATOM 345 CB SER A 40 12.721 37.433 3.889 1.00 17.10 ATOM 346 OG SER A 40 12.109 37.609 2.621 1.00 17.63 ATOM 347 C SER A 40 14.804 36.625 4.965 1.00 16.18 ATOM 348 O SER A 40 14.580 35.489 5.400 1.00 16.02 ATOM 349 N PRO A 41 15.636 37.477 5.538 1.00 16.16 ATOM 350 CA PRO A 41 16.177 37.231 6.876 1.00 15.95 ATOM 351 CB PRO A 41 17.273 38.241 7.028 1.00 16.47 ATOM 352 CG PRO A 41 17.185 39.250 5.894 1.00 16.77 ATOM 353 CD PRO A 41 16.067 38.763 4.989 1.00 16.71 ATOM 354 C PRO A 41 15.114 37.434 7.957 1.00 15.51 ATOM 355 O PRO A 41 14.924 38.547 8.457 1.00 15.39 ATOM 356 N ALA A 42 14.424 36.364 8.300 1.00 15.56 ATOM 358 CA ALA A 42 13.462 36.422 9.397 1.00 15.32 ATOM 359 CB ALA A 42 12.173 35.720 8.986 1.00 15.56 ATOM 360 C ALA A 42 14.074 35.757 10.620 1.00 15.35 ATOM 361 O ALA A 42 15.113 36.201 11.123 1.00 15.57 ATOM 362 N ASN A 43 13.442 34.692 11.080 1.00 15.19 ATOM 364 CA ASN A 43 13.969 33.959 12.235 1.00 15.22 ATOM 365 CB ASN A 43 12.840 33.244 12.961 1.00 15.86 ATOM 366 CG ASN A 43 11.718 34.223 13.290 1.00 15.73 ATOM 367 OD1 ASN A 43 11.957 35.352 13.733 1.00 15.55 ATOM 368 ND2 ASN A 43 10.500 33.783 13.031 1.00 15.89 ATOM 371 C ASN A 43 15.024 32.956 11.791 1.00 15.19 ATOM 372 O ASN A 43 14.746 31.882 11.237 1.00 14.96 ATOM 373 N ALA A 44 16.258 33.361 12.016 1.00 15.41 ATOM 375 CA ALA A 44 17.414 32.551 11.648 1.00 15.49 ATOM 376 CB ALA A 44 18.618 33.479 11.534 1.00 16.78 ATOM 377 C ALA A 44 17.689 31.461 12.680 1.00 14.95 ATOM 378 O ALA A 44 17.239 31.539 13.828 1.00 14.99 ATOM 379 N PRO A 45 18.323 30.397 12.217 1.00 14.51 ATOM 380 CA PRO A 45 18.417 30.065 10.794 1.00 14.09 ATOM 381 CB PRO A 45 19.749 29.386 10.732 1.00 15.04 ATOM 382 CG PRO A 45 19.991 28.754 12.102 1.00 15.75 ATOM 383 CD PRO A 45 18.914 29.328 13.016 1.00 15.56 ATOM 384 C PRO A 45 17.366 29.040 10.370 1.00 13.14 ATOM 385 O PRO A 45 17.726 28.150 9.594 1.00 12.61 ATOM 386 N SER A 46 16.093 29.241 10.684 1.00 12.99 ATOM 388 CA SER A 46 15.135 28.120 10.687 1.00 12.17 ATOM 389 CB SER A 46 13.896 28.570 11.446 1.00 12.36 ATOM 390 OG SER A 46 14.304 29.125 12.688 1.00 13.04 ATOM 391 C SER A 46 14.710 27.579 9.315 1.00 11.42 ATOM 392 O SER A 46 14.251 26.433 9.244 1.00 10.99 ATOM 393 N ALA A 47 15.008 28.290 8.241 1.00 11.41 ATOM 395 CA ALA A 47 14.719 27.767 6.902 1.00 10.80 ATOM 396 CB ALA A 47 14.344 28.923 5.995 1.00 11.50 ATOM 397 C ALA A 47 15.892 26.992 6.294 1.00 10.75 ATOM 398 O ALA A 47 15.772 26.447 5.193 1.00 10.39 ATOM 399 N TYR A 48 17.013 26.957 6.995 1.00 12.04 ATOM 401 CA TYR A 48 18.177 26.175 6.553 1.00 11.89 ATOM 402 CB TYR A 48 19.448 26.926 6.935 1.00 12.88 ATOM 403 CG TYR A 48 20.719 26.229 6.463 1.00 13.41 ATOM 404 CD1 TYR A 48 20.842 25.842 5.134 1.00 13.91 ATOM 405 CE1 TYR A 48 21.991 25.194 4.703 1.00 14.50 ATOM 406 CZ TYR A 48 23.016 24.938 5.603 1.00 14.60 ATOM 407 OH TYR A 48 24.103 24.194 5.203 1.00 15.31 ATOM 408 CE2 TYR A 48 22.904 25.338 6.929 1.00 14.16 ATOM 409 CD2 TYR A 48 21.753 25.985 7.359 1.00 13.55 ATOM 410 C TYR A 48 18.251 24.717 7.075 1.00 11.54 ATOM 411 O TYR A 48 18.628 23.860 6.261 1.00 11.71 ATOM 412 N PRO A 49 17.912 24.374 8.323 1.00 11.19 ATOM 413 CA PRO A 49 17.932 22.951 8.679 1.00 10.95 ATOM 414 CB PRO A 49 17.660 22.880 10.145 1.00 11.17 ATOM 415 CG PRO A 49 17.388 24.271 10.678 1.00 11.31 ATOM 416 CD PRO A 49 17.545 25.195 9.489 1.00 11.40 ATOM 417 C PRO A 49 16.905 22.114 7.930 1.00 10.04 ATOM 418 O PRO A 49 17.225 20.968 7.584 1.00 9.90 ATOM 419 N ARG A 50 15.827 22.740 7.482 1.00 9.55 ATOM 421 CA ARG A 50 14.794 22.014 6.748 1.00 8.74 ATOM 422 CB ARG A 50 13.594 22.910 6.472 1.00 9.11 ATOM 423 CG ARG A 50 12.455 22.038 5.975 1.00 9.19 ATOM 424 CD ARG A 50 12.107 21.007 7.043 1.00 9.81 ATOM 425 NE ARG A 50 11.247 19.944 6.506 1.00 10.42 ATOM 426 CZ ARG A 50 10.609 19.062 7.276 1.00 11.31 ATOM 427 NH1 ARG A 50 10.725 19.129 8.604 1.00 12.00 ATOM 428 NH2 ARG A 50 9.837 18.127 6.718 1.00 11.69 ATOM 429 C ARG A 50 15.308 21.335 5.466 1.00 8.18 ATOM 430 O ARG A 50 15.260 20.103 5.438 1.00 8.13 ATOM 431 N PRO A 51 15.873 22.027 4.482 1.00 7.98 ATOM 432 CA PRO A 51 16.236 21.311 3.255 1.00 7.63 ATOM 433 CB PRO A 51 16.414 22.386 2.227 1.00 8.38 ATOM 434 CG PRO A 51 16.443 23.742 2.908 1.00 9.01 ATOM 435 CD PRO A 51 16.118 23.472 4.362 1.00 8.97 ATOM 436 C PRO A 51 17.507 20.444 3.310 1.00 7.66 ATOM 437 O PRO A 51 17.716 19.692 2.350 1.00 7.43 ATOM 438 N SER A 52 18.302 20.463 4.373 1.00 8.11 ATOM 440 CA SER A 52 19.584 19.749 4.262 1.00 8.31 ATOM 441 CB SER A 52 20.475 20.588 3.351 1.00 9.41 ATOM 442 OG SER A 52 20.648 21.850 3.986 1.00 10.16 ATOM 443 C SER A 52 20.376 19.533 5.550 1.00 7.49 ATOM 444 O SER A 52 21.315 18.726 5.531 1.00 7.60 ATOM 445 N SER A 53 20.008 20.183 6.640 1.00 6.82 ATOM 447 CA SER A 53 20.992 20.341 7.719 1.00 6.11 ATOM 448 CB SER A 53 21.021 21.802 8.132 1.00 8.00 ATOM 449 OG SER A 53 21.153 22.562 6.938 1.00 8.48 ATOM 450 C SER A 53 20.803 19.469 8.954 1.00 5.80 ATOM 451 O SER A 53 19.680 19.110 9.342 1.00 6.02 ATOM 452 N THR A 54 21.872 19.538 9.736 1.00 6.64 ATOM 454 CA THR A 54 22.105 18.732 10.950 1.00 6.72 ATOM 455 CB THR A 54 23.588 18.765 11.298 1.00 7.78 ATOM 456 OG1 THR A 54 23.918 20.088 11.700 1.00 8.09 ATOM 457 CG2 THR A 54 24.477 18.369 10.126 1.00 8.15 ATOM 458 C THR A 54 21.346 19.183 12.197 1.00 6.30 ATOM 459 O THR A 54 21.670 18.742 13.305 1.00 6.19 ATOM 460 N LYS A 55 20.346 20.027 12.031 1.00 6.22 ATOM 462 CA LYS A 55 19.660 20.554 13.200 1.00 6.00 ATOM 463 CB LYS A 55 19.417 22.045 12.977 1.00 6.38 ATOM 464 CG LYS A 55 18.813 22.772 14.181 1.00 7.21 ATOM 465 CD LYS A 55 19.856 23.354 15.142 1.00 7.56 ATOM 466 CE LYS A 55 20.675 22.295 15.874 1.00 8.08 ATOM 467 NZ LYS A 55 21.674 22.919 16.753 1.00 8.26 ATOM 468 C LYS A 55 18.341 19.833 13.484 1.00 5.00 ATOM 469 O LYS A 55 17.994 19.685 14.661 1.00 5.17 ATOM 470 N SER A 56 17.660 19.312 12.470 1.00 4.28 ATOM 472 CA SER A 56 16.344 18.717 12.756 1.00 3.33 ATOM 473 CB SER A 56 15.358 19.866 12.979 1.00 2.95 ATOM 474 OG SER A 56 15.435 20.755 11.867 1.00 3.03 ATOM 475 C SER A 56 15.751 17.768 11.705 1.00 2.29 ATOM 476 O SER A 56 15.363 16.644 12.058 1.00 2.75 ATOM 477 N THR A 57 15.867 18.108 10.433 1.00 1.18 ATOM 479 CA THR A 57 14.941 17.542 9.435 1.00 0.39 ATOM 480 CB THR A 57 15.098 18.307 8.142 1.00 1.62 ATOM 481 OG1 THR A 57 14.781 19.647 8.463 1.00 2.31 ATOM 482 CG2 THR A 57 14.113 17.803 7.097 1.00 2.10 ATOM 483 C THR A 57 14.971 16.022 9.224 1.00 0.19 ATOM 484 O THR A 57 13.954 15.407 9.576 1.00 0.28 ATOM 485 N PRO A 58 16.067 15.391 8.806 1.00 0.34 ATOM 486 CA PRO A 58 16.016 13.941 8.552 1.00 0.33 ATOM 487 CB PRO A 58 17.316 13.606 7.896 1.00 1.25 ATOM 488 CG PRO A 58 18.167 14.854 7.779 1.00 1.43 ATOM 489 CD PRO A 58 17.362 15.969 8.412 1.00 1.02 ATOM 490 C PRO A 58 15.748 13.029 9.760 1.00 0.16 ATOM 491 O PRO A 58 15.268 11.914 9.523 1.00 0.19 ATOM 492 N ALA A 59 15.759 13.536 10.987 1.00 0.18 ATOM 494 CA ALA A 59 15.399 12.691 12.132 1.00 0.16 ATOM 495 CB ALA A 59 15.816 13.375 13.426 1.00 0.63 ATOM 496 C ALA A 59 13.893 12.442 12.176 1.00 0.16 ATOM 497 O ALA A 59 13.474 11.296 12.374 1.00 0.31 ATOM 498 N SER A 60 13.125 13.402 11.685 1.00 0.01 ATOM 500 CA SER A 60 11.671 13.222 11.618 1.00 0.01 ATOM 501 CB SER A 60 10.987 14.584 11.676 1.00 0.23 ATOM 502 OG SER A 60 11.286 15.282 10.474 1.00 1.03 ATOM 503 C SER A 60 11.255 12.503 10.338 1.00 0.00 ATOM 504 O SER A 60 10.154 11.946 10.267 1.00 0.01 ATOM 505 N GLN A 61 12.183 12.377 9.404 1.00 0.01 ATOM 507 CA GLN A 61 11.887 11.693 8.150 1.00 0.00 ATOM 508 CB GLN A 61 12.694 12.362 7.050 1.00 0.37 ATOM 509 CG GLN A 61 12.342 13.843 6.975 1.00 1.02 ATOM 510 CD GLN A 61 13.163 14.531 5.893 1.00 1.17 ATOM 511 OE1 GLN A 61 14.396 14.598 5.976 1.00 1.88 ATOM 512 NE2 GLN A 61 12.459 15.114 4.939 1.00 1.51 ATOM 515 C GLN A 61 12.235 10.212 8.246 1.00 0.01 ATOM 516 O GLN A 61 11.542 9.376 7.652 1.00 0.01 ATOM 517 N VAL A 62 13.136 9.883 9.160 1.00 0.00 ATOM 519 CA VAL A 62 13.470 8.477 9.399 1.00 0.01 ATOM 520 CB VAL A 62 14.932 8.330 9.823 1.00 0.37 ATOM 521 CG1 VAL A 62 15.873 8.901 8.768 1.00 0.73 ATOM 522 CG2 VAL A 62 15.210 8.953 11.185 1.00 0.81 ATOM 523 C VAL A 62 12.547 7.846 10.441 1.00 0.01 ATOM 524 O VAL A 62 12.561 6.617 10.582 1.00 0.01 ATOM 525 N TYR A 63 11.609 8.627 10.964 1.00 0.00 ATOM 527 CA TYR A 63 10.620 8.144 11.940 1.00 0.01 ATOM 528 CB TYR A 63 9.681 9.308 12.252 1.00 0.95 ATOM 529 CG TYR A 63 8.348 8.934 12.904 1.00 1.26 ATOM 530 CD1 TYR A 63 7.161 9.196 12.228 1.00 1.76 ATOM 531 CE1 TYR A 63 5.941 8.859 12.802 1.00 2.50 ATOM 532 CZ TYR A 63 5.910 8.263 14.056 1.00 2.77 ATOM 533 OH TYR A 63 4.700 7.893 14.609 1.00 3.61 ATOM 534 CE2 TYR A 63 7.092 8.010 14.738 1.00 2.43 ATOM 535 CD2 TYR A 63 8.311 8.346 14.163 1.00 1.69 ATOM 536 C TYR A 63 9.786 6.981 11.420 1.00 0.01 ATOM 537 O TYR A 63 9.729 5.938 12.085 1.00 0.00 ATOM 538 N SER A 64 9.388 7.048 10.160 1.00 0.00 ATOM 540 CA SER A 64 8.518 6.008 9.618 1.00 0.01 ATOM 541 CB SER A 64 7.937 6.501 8.301 1.00 0.07 ATOM 542 OG SER A 64 7.191 7.679 8.576 1.00 0.58 ATOM 543 C SER A 64 9.271 4.708 9.388 1.00 0.00 ATOM 544 O SER A 64 8.802 3.659 9.844 1.00 0.01 ATOM 545 N LEU A 65 10.541 4.814 9.043 1.00 0.01 ATOM 547 CA LEU A 65 11.302 3.606 8.729 1.00 0.00 ATOM 548 CB LEU A 65 12.391 3.989 7.736 1.00 0.19 ATOM 549 CG LEU A 65 11.769 4.693 6.527 1.00 0.31 ATOM 550 CD1 LEU A 65 12.829 5.223 5.568 1.00 0.42 ATOM 551 CD2 LEU A 65 10.776 3.799 5.787 1.00 0.30 ATOM 552 C LEU A 65 11.868 2.975 9.999 1.00 0.00 ATOM 553 O LEU A 65 11.751 1.751 10.154 1.00 0.01 ATOM 554 N ASN A 66 12.057 3.813 11.008 1.00 0.01 ATOM 556 CA ASN A 66 12.478 3.354 12.336 1.00 0.01 ATOM 557 CB ASN A 66 12.838 4.583 13.174 1.00 0.20 ATOM 558 CG ASN A 66 13.358 4.202 14.562 1.00 0.42 ATOM 559 OD1 ASN A 66 14.527 3.832 14.714 1.00 1.44 ATOM 560 ND2 ASN A 66 12.506 4.348 15.567 1.00 0.90 ATOM 563 C ASN A 66 11.341 2.604 13.017 1.00 0.01 ATOM 564 O ASN A 66 11.543 1.472 13.473 1.00 0.01 ATOM 565 N THR A 67 10.125 3.104 12.857 1.00 0.00 ATOM 567 CA THR A 67 8.970 2.428 13.458 1.00 0.00 ATOM 568 CB THR A 67 7.800 3.398 13.595 1.00 0.32 ATOM 569 OG1 THR A 67 7.469 3.918 12.315 1.00 1.02 ATOM 570 CG2 THR A 67 8.152 4.565 14.508 1.00 1.22 ATOM 571 C THR A 67 8.546 1.202 12.655 1.00 0.01 ATOM 572 O THR A 67 8.146 0.206 13.268 1.00 0.00 ATOM 573 N ASP A 68 8.887 1.166 11.376 1.00 0.01 ATOM 575 CA ASP A 68 8.646 −0.036 10.573 1.00 0.01 ATOM 576 CB ASP A 68 8.899 0.265 9.098 1.00 0.09 ATOM 577 CG ASP A 68 7.881 1.263 8.547 1.00 0.60 ATOM 578 OD1 ASP A 68 6.748 1.239 9.009 1.00 0.78 ATOM 579 OD2 ASP A 68 8.223 1.944 7.587 1.00 1.02 ATOM 580 C ASP A 68 9.571 −1.160 11.021 1.00 0.01 ATOM 581 O ASP A 68 9.068 −2.216 11.426 1.00 0.01 ATOM 582 N PHE A 69 10.832 −0.827 11.256 1.00 0.00 ATOM 584 CA PHE A 69 11.808 −1.808 11.748 1.00 0.01 ATOM 585 CB PHE A 69 13.193 −1.172 11.652 1.00 0.01 ATOM 586 CG PHE A 69 14.294 −1.923 12.397 1.00 0.01 ATOM 587 CD1 PHE A 69 14.711 −3.173 11.958 1.00 0.00 ATOM 588 CE1 PHE A 69 15.708 −3.853 12.645 1.00 0.01 ATOM 589 CZ PHE A 69 16.288 −3.285 13.772 1.00 0.01 ATOM 590 CE2 PHE A 69 15.871 −2.035 14.212 1.00 0.01 ATOM 591 CD2 PHE A 69 14.874 −1.355 13.525 1.00 0.01 ATOM 592 C PHE A 69 11.537 −2.226 13.195 1.00 0.01 ATOM 593 O PHE A 69 11.671 −3.414 13.524 1.00 0.01 ATOM 594 N ALA A 70 10.940 −1.334 13.966 1.00 0.01 ATOM 596 CA ALA A 70 10.573 −1.645 15.346 1.00 0.01 ATOM 597 CB ALA A 70 10.227 −0.336 16.042 1.00 0.01 ATOM 598 C ALA A 70 9.382 −2.598 15.426 1.00 0.00 ATOM 599 O ALA A 70 9.438 −3.570 16.190 1.00 0.01 ATOM 600 N PHE A 71 8.457 −2.481 14.485 1.00 0.01 ATOM 602 CA PHE A 71 7.313 −3.398 14.453 1.00 0.01 ATOM 603 CB PHE A 71 6.218 −2.830 13.556 1.00 0.00 ATOM 604 CG PHE A 71 5.568 −1.540 14.048 1.00 0.01 ATOM 605 CD1 PHE A 71 5.361 −1.330 15.406 1.00 0.01 ATOM 606 CE1 PHE A 71 4.772 −0.151 15.843 1.00 0.00 ATOM 607 CZ PHE A 71 4.383 0.813 14.922 1.00 0.00 ATOM 608 CE2 PHE A 71 4.575 0.596 13.564 1.00 0.00 ATOM 609 CD2 PHE A 71 5.163 −0.583 13.127 1.00 0.00 ATOM 610 C PHE A 71 7.731 −4.758 13.915 1.00 0.00 ATOM 611 O PHE A 71 7.347 −5.783 14.495 1.00 0.01 ATOM 612 N ARG A 72 8.702 −4.756 13.016 1.00 0.00 ATOM 614 CA ARG A 72 9.225 −6.011 12.474 1.00 0.01 ATOM 615 CB ARG A 72 10.216 −5.697 11.365 1.00 0.20 ATOM 616 CG ARG A 72 9.590 −4.923 10.217 1.00 0.96 ATOM 617 CD ARG A 72 10.667 −4.523 9.219 1.00 1.05 ATOM 618 NE ARG A 72 10.152 −3.580 8.218 1.00 1.27 ATOM 619 CZ ARG A 72 10.946 −3.002 7.317 1.00 1.66 ATOM 620 NH1 ARG A 72 10.449 −2.099 6.470 1.00 2.10 ATOM 621 NH2 ARG A 72 12.249 −3.292 7.300 1.00 2.00 ATOM 622 C ARG A 72 9.964 −6.806 13.537 1.00 0.01 ATOM 623 O ARG A 72 9.629 −7.974 13.760 1.00 0.00 ATOM 624 N LEU A 73 10.764 −6.124 14.341 1.00 0.00 ATOM 626 CA LEU A 73 11.537 −6.823 15.365 1.00 0.01 ATOM 627 CB LEU A 73 12.664 −5.899 15.815 1.00 0.01 ATOM 628 CG LEU A 73 13.628 −6.598 16.766 1.00 0.01 ATOM 629 CD1 LEU A 73 14.126 −7.909 16.170 1.00 0.01 ATOM 630 CD2 LEU A 73 14.801 −5.690 17.118 1.00 0.01 ATOM 631 C LEU A 73 10.670 −7.237 16.555 1.00 0.01 ATOM 632 O LEU A 73 10.824 −8.367 17.037 1.00 0.01 ATOM 633 N TYR A 74 9.610 −6.490 16.824 1.00 0.01 ATOM 635 CA TYR A 74 8.727 −6.861 17.930 1.00 0.01 ATOM 636 CB TYR A 74 7.800 −5.698 18.262 1.00 0.01 ATOM 637 CG TYR A 74 6.878 −5.984 19.444 1.00 0.01 ATOM 638 CD1 TYR A 74 7.378 −5.918 20.739 1.00 0.01 ATOM 639 CE1 TYR A 74 6.547 −6.185 21.819 1.00 0.01 ATOM 640 CZ TYR A 74 5.217 −6.518 21.600 1.00 0.00 ATOM 641 OH TYR A 74 4.393 −6.794 22.670 1.00 0.01 ATOM 642 CE2 TYR A 74 4.714 −6.584 20.307 1.00 0.01 ATOM 643 CD2 TYR A 74 5.546 −6.316 19.228 1.00 0.01 ATOM 644 C TYR A 74 7.896 −8.082 17.574 1.00 0.01 ATOM 645 O TYR A 74 7.932 −9.072 18.316 1.00 0.01 ATOM 646 N ARG A 75 7.412 −8.126 16.344 1.00 0.01 ATOM 648 CA ARG A 75 6.597 −9.263 15.920 1.00 0.00 ATOM 649 CB ARG A 75 5.781 −8.854 14.703 1.00 0.00 ATOM 650 CG ARG A 75 4.855 −7.695 15.048 1.00 0.01 ATOM 651 CD ARG A 75 4.096 −7.205 13.823 1.00 0.00 ATOM 652 NE ARG A 75 3.269 −6.038 14.165 1.00 0.00 ATOM 653 CZ ARG A 75 3.021 −5.040 13.315 1.00 0.01 ATOM 654 NH1 ARG A 75 3.530 −5.072 12.082 1.00 0.00 ATOM 655 NH2 ARG A 75 2.263 −4.010 13.697 1.00 0.01 ATOM 656 C ARG A 75 7.456 −10.480 15.599 1.00 0.01 ATOM 657 O ARG A 75 7.014 −11.604 15.862 1.00 0.00 ATOM 658 N ARG A 76 8.734 −10.261 15.336 1.00 0.01 ATOM 660 CA ARG A 76 9.650 −11.381 15.129 1.00 0.01 ATOM 661 CB ARG A 76 10.935 −10.852 14.504 1.00 0.28 ATOM 662 CG ARG A 76 11.790 −11.993 13.973 1.00 1.10 ATOM 663 CD ARG A 76 11.014 −12.768 12.914 1.00 1.58 ATOM 664 NE ARG A 76 11.815 −13.856 12.336 1.00 2.21 ATOM 665 CZ ARG A 76 11.783 −14.161 11.037 1.00 2.96 ATOM 666 NH1 ARG A 76 12.433 −15.236 10.589 1.00 3.85 ATOM 667 NH2 ARG A 76 11.019 −13.449 10.206 1.00 3.23 ATOM 668 C ARG A 76 9.975 −12.057 16.456 1.00 0.01 ATOM 669 O ARG A 76 9.903 −13.288 16.537 1.00 0.01 ATOM 670 N LEU A 77 10.031 −11.279 17.525 1.00 0.01 ATOM 672 CA LEU A 77 10.285 −11.858 18.848 1.00 0.01 ATOM 673 CB LEU A 77 10.772 −10.752 19.775 1.00 0.01 ATOM 674 CG LEU A 77 12.100 −10.176 19.297 1.00 0.01 ATOM 675 CD1 LEU A 77 12.468 −8.915 20.069 1.00 0.01 ATOM 676 CD2 LEU A 77 13.216 −11.213 19.375 1.00 0.01 ATOM 677 C LEU A 77 9.033 −12.517 19.425 1.00 0.01 ATOM 678 O LEU A 77 9.147 −13.600 20.020 1.00 0.01 ATOM 679 N VAL A 78 7.869 −12.043 19.002 1.00 0.01 ATOM 681 CA VAL A 78 6.603 −12.672 19.402 1.00 0.01 ATOM 682 CB VAL A 78 5.447 −11.736 19.045 1.00 0.11 ATOM 683 CG1 VAL A 78 4.095 −12.384 19.325 1.00 0.20 ATOM 684 CG2 VAL A 78 5.551 −10.403 19.773 1.00 0.20 ATOM 685 C VAL A 78 6.398 −13.999 18.675 1.00 0.01 ATOM 686 O VAL A 78 6.015 −14.989 19.310 1.00 0.01 ATOM 687 N LEU A 79 6.872 −14.068 17.439 1.00 0.01 ATOM 689 CA LEU A 79 6.763 −15.296 16.644 1.00 0.01 ATOM 690 CB LEU A 79 6.776 −14.925 15.166 1.00 0.19 ATOM 691 CG LEU A 79 5.533 −14.129 14.784 1.00 0.24 ATOM 692 CD1 LEU A 79 5.635 −13.603 13.357 1.00 0.38 ATOM 693 CD2 LEU A 79 4.266 −14.957 14.971 1.00 0.37 ATOM 694 C LEU A 79 7.882 −16.294 16.933 1.00 0.01 ATOM 695 O LEU A 79 7.786 −17.458 16.528 1.00 0.01 ATOM 696 N GLU A 80 8.912 −15.863 17.642 1.00 0.01 ATOM 698 CA GLU A 80 9.894 −16.823 18.142 1.00 0.01 ATOM 699 CB GLU A 80 11.209 −16.127 18.478 1.00 0.49 ATOM 700 CG GLU A 80 11.857 −15.481 17.258 1.00 1.39 ATOM 701 CD GLU A 80 12.079 −16.498 16.142 1.00 1.48 ATOM 702 OE1 GLU A 80 11.433 −16.347 15.114 1.00 2.26 ATOM 703 OE2 GLU A 80 13.034 −17.252 16.255 1.00 1.23 ATOM 704 C GLU A 80 9.318 −17.467 19.394 1.00 0.01 ATOM 705 O GLU A 80 9.257 −18.698 19.495 1.00 0.01 ATOM 706 N THR A 81 8.856 −16.619 20.301 1.00 0.01 ATOM 708 CA THR A 81 8.132 −17.067 21.498 1.00 0.02 ATOM 709 CB THR A 81 8.969 −17.980 22.395 1.00 1.52 ATOM 710 OG1 THR A 81 8.309 −18.010 23.652 1.00 2.01 ATOM 711 CG2 THR A 81 10.389 −17.486 22.633 1.00 2.60 ATOM 712 C THR A 81 7.596 −15.897 22.316 1.00 0.01 ATOM 713 O THR A 81 8.349 −15.135 22.945 1.00 0.01 ATOM 714 N PRO A 82 6.281 −15.924 22.474 1.00 0.01 ATOM 715 CA PRO A 82 5.547 −14.945 23.285 1.00 0.01 ATOM 716 CB PRO A 82 4.116 −15.121 22.876 1.00 0.18 ATOM 717 CG PRO A 82 3.983 −16.396 22.058 1.00 0.19 ATOM 718 CD PRO A 82 5.392 −16.932 21.889 1.00 0.14 ATOM 719 C PRO A 82 5.680 −15.115 24.809 1.00 0.01 ATOM 720 O PRO A 82 5.041 −14.357 25.547 1.00 0.01 ATOM 721 N SER A 83 6.493 −16.055 25.281 1.00 0.02 ATOM 723 CA SER A 83 6.697 −16.235 26.722 1.00 0.01 ATOM 724 CB SER A 83 6.985 −17.702 27.015 1.00 0.01 ATOM 725 OG SER A 83 8.279 −18.000 26.509 1.00 0.02 ATOM 726 C SER A 83 7.868 −15.393 27.232 1.00 0.01 ATOM 727 O SER A 83 8.277 −15.528 28.391 1.00 0.01 ATOM 728 N GLN A 84 8.492 −14.651 26.334 1.00 0.02 ATOM 730 CA GLN A 84 9.553 −13.730 26.735 1.00 0.01 ATOM 731 CB GLN A 84 10.513 −13.545 25.567 1.00 1.11 ATOM 732 CG GLN A 84 11.173 −14.843 25.130 1.00 1.61 ATOM 733 CD GLN A 84 12.060 −14.558 23.922 1.00 1.93 ATOM 734 OE1 GLN A 84 13.283 −14.730 23.977 1.00 2.66 ATOM 735 NE2 GLN A 84 11.422 −14.146 22.838 1.00 1.90 ATOM 738 C GLN A 84 8.991 −12.358 27.076 1.00 0.00 ATOM 739 O GLN A 84 8.119 −11.841 26.368 1.00 0.01 ATOM 740 N ASN A 85 9.526 −11.766 28.132 1.00 0.01 ATOM 742 CA ASN A 85 9.314 −10.334 28.379 1.00 0.01 ATOM 743 CB ASN A 85 9.736 −9.974 29.798 1.00 0.01 ATOM 744 CG ASN A 85 8.816 −10.618 30.824 1.00 0.02 ATOM 745 OD1 ASN A 85 7.585 −10.488 30.751 1.00 0.01 ATOM 746 ND2 ASN A 85 9.426 −11.245 31.811 1.00 0.01 ATOM 749 C ASN A 85 10.170 −9.542 27.399 1.00 0.01 ATOM 750 O ASN A 85 11.369 −9.336 27.617 1.00 0.01 ATOM 751 N ILE A 86 9.554 −9.156 26.297 1.00 0.01 ATOM 753 CA ILE A 86 10.277 −8.469 25.226 1.00 0.01 ATOM 754 CB ILE A 86 9.369 −8.454 23.996 1.00 0.01 ATOM 755 CG2 ILE A 86 10.021 −7.781 22.792 1.00 0.00 ATOM 756 CG1 ILE A 86 8.974 −9.879 23.629 1.00 0.01 ATOM 757 CD1 ILE A 86 8.153 −9.905 22.347 1.00 0.01 ATOM 758 C ILE A 86 10.661 −7.064 25.671 1.00 0.00 ATOM 759 O ILE A 86 9.906 −6.417 26.406 1.00 0.01 ATOM 760 N PHE A 87 11.877 −6.664 25.342 1.00 0.01 ATOM 762 CA PHE A 87 12.324 −5.303 25.630 1.00 0.00 ATOM 763 CB PHE A 87 12.633 −5.134 27.110 1.00 0.01 ATOM 764 CG PHE A 87 12.846 −3.664 27.423 1.00 0.01 ATOM 765 CD1 PHE A 87 11.782 −2.784 27.282 1.00 0.00 ATOM 766 CE1 PHE A 87 11.964 −1.432 27.531 1.00 0.01 ATOM 767 CZ PHE A 87 13.213 −0.964 27.912 1.00 0.01 ATOM 768 CE2 PHE A 87 14.278 −1.844 28.051 1.00 0.00 ATOM 769 CD2 PHE A 87 14.094 −3.197 27.807 1.00 0.01 ATOM 770 C PHE A 87 13.559 −4.950 24.810 1.00 0.01 ATOM 771 O PHE A 87 14.695 −5.254 25.195 1.00 0.01 ATOM 772 N PHE A 88 13.324 −4.284 23.696 1.00 0.01 ATOM 774 CA PHE A 88 14.428 −3.886 22.819 1.00 0.01 ATOM 775 CB PHE A 88 14.399 −4.710 21.532 1.00 0.01 ATOM 776 CG PHE A 88 13.216 −4.453 20.600 1.00 0.01 ATOM 777 CD1 PHE A 88 12.037 −5.171 20.752 1.00 0.01 ATOM 778 CE1 PHE A 88 10.966 −4.930 19.904 1.00 0.01 ATOM 779 CZ PHE A 88 11.076 −3.979 18.898 1.00 0.01 ATOM 780 CE2 PHE A 88 12.258 −3.272 18.735 1.00 0.01 ATOM 781 CD2 PHE A 88 13.329 −3.513 19.583 1.00 0.01 ATOM 782 C PHE A 88 14.369 −2.404 22.477 1.00 0.00 ATOM 783 O PHE A 88 13.301 −1.779 22.467 1.00 0.01 ATOM 784 N SER A 89 15.537 −1.856 22.202 1.00 0.01 ATOM 786 CA SER A 89 15.633 −0.485 21.714 1.00 0.00 ATOM 787 CB SER A 89 16.812 0.213 22.369 1.00 0.01 ATOM 788 OG SER A 89 17.000 1.443 21.675 1.00 0.01 ATOM 789 C SER A 89 15.843 −0.446 20.212 1.00 0.01 ATOM 790 O SER A 89 16.975 −0.633 19.739 1.00 0.01 ATOM 791 N PRO A 90 14.804 −0.047 19.493 1.00 0.01 ATOM 792 CA PRO A 90 14.926 0.148 18.048 1.00 0.01 ATOM 793 CB PRO A 90 13.554 0.526 17.581 1.00 0.01 ATOM 794 CG PRO A 90 12.621 0.615 18.780 1.00 0.01 ATOM 795 CD PRO A 90 13.465 0.275 19.995 1.00 0.01 ATOM 796 C PRO A 90 15.947 1.233 17.718 1.00 0.01 ATOM 797 O PRO A 90 16.867 0.951 16.942 1.00 0.01 ATOM 798 N VAL A 91 15.995 2.264 18.549 1.00 0.00 ATOM 800 CA VAL A 91 16.952 3.354 18.363 1.00 0.01 ATOM 801 CB VAL A 91 16.642 4.444 19.381 1.00 0.01 ATOM 802 CG1 VAL A 91 17.798 5.427 19.540 1.00 0.01 ATOM 803 CG2 VAL A 91 15.361 5.174 19.001 1.00 0.01 ATOM 804 C VAL A 91 18.409 2.908 18.486 1.00 0.01 ATOM 805 O VAL A 91 19.147 3.108 17.515 1.00 0.00 ATOM 806 N SER A 92 18.764 2.110 19.485 1.00 0.01 ATOM 808 CA SER A 92 20.179 1.744 19.608 1.00 0.01 ATOM 809 CB SER A 92 20.433 1.193 21.006 1.00 0.01 ATOM 810 OG SER A 92 19.684 −0.004 21.158 1.00 0.01 ATOM 811 C SER A 92 20.633 0.734 18.551 1.00 0.01 ATOM 812 O SER A 92 21.717 0.937 17.981 1.00 0.01 ATOM 813 N VAL A 93 19.729 −0.115 18.086 1.00 0.00 ATOM 815 CA VAL A 93 20.106 −1.096 17.067 1.00 0.01 ATOM 816 CB VAL A 93 19.066 −2.211 17.045 1.00 0.09 ATOM 817 CG1 VAL A 93 19.417 −3.261 15.997 1.00 0.13 ATOM 818 CG2 VAL A 93 18.926 −2.862 18.413 1.00 0.17 ATOM 819 C VAL A 93 20.176 −0.450 15.689 1.00 0.00 ATOM 820 O VAL A 93 21.195 −0.594 15.001 1.00 0.01 ATOM 821 N SER A 94 19.274 0.482 15.434 1.00 0.01 ATOM 823 CA SER A 94 19.235 1.133 14.121 1.00 0.01 ATOM 824 CB SER A 94 17.859 1.765 13.922 1.00 0.17 ATOM 825 OG SER A 94 17.660 2.767 14.914 1.00 0.70 ATOM 826 C SER A 94 20.327 2.189 13.969 1.00 0.01 ATOM 827 O SER A 94 20.922 2.291 12.888 1.00 0.01 ATOM 828 N THR A 95 20.775 2.742 15.084 1.00 0.01 ATOM 830 CA THR A 95 21.859 3.719 15.038 1.00 0.00 ATOM 831 CB THR A 95 21.807 4.547 16.318 1.00 0.01 ATOM 832 OG1 THR A 95 20.577 5.260 16.317 1.00 0.02 ATOM 833 CG2 THR A 95 22.937 5.566 16.388 1.00 0.01 ATOM 834 C THR A 95 23.211 3.027 14.903 1.00 0.01 ATOM 835 O THR A 95 24.026 3.454 14.076 1.00 0.00 ATOM 836 N SER A 96 23.321 1.827 15.451 1.00 0.01 ATOM 838 CA SER A 96 24.582 1.089 15.319 1.00 0.01 ATOM 839 CB SER A 96 24.727 0.089 16.462 1.00 0.15 ATOM 840 OG SER A 96 23.632 −0.817 16.438 1.00 0.84 ATOM 841 C SER A 96 24.688 0.379 13.970 1.00 0.01 ATOM 842 O SER A 96 25.791 0.294 13.416 1.00 0.01 ATOM 843 N LEU A 97 23.559 0.119 13.333 1.00 0.01 ATOM 845 CA LEU A 97 23.607 −0.467 11.994 1.00 0.01 ATOM 846 CB LEU A 97 22.315 −1.224 11.738 1.00 0.13 ATOM 847 CG LEU A 97 22.233 −2.463 12.617 1.00 0.13 ATOM 848 CD1 LEU A 97 20.874 −3.135 12.478 1.00 0.64 ATOM 849 CD2 LEU A 97 23.361 −3.436 12.290 1.00 0.47 ATOM 850 C LEU A 97 23.828 0.591 10.918 1.00 0.00 ATOM 851 O LEU A 97 24.610 0.347 9.990 1.00 0.00 ATOM 852 N ALA A 98 23.405 1.816 11.182 1.00 0.01 ATOM 854 CA ALA A 98 23.698 2.904 10.243 1.00 0.01 ATOM 855 CB ALA A 98 22.655 3.993 10.410 1.00 0.01 ATOM 856 C ALA A 98 25.103 3.470 10.455 1.00 0.00 ATOM 857 O ALA A 98 25.659 4.131 9.572 1.00 0.01 ATOM 858 N MET A 99 25.702 3.114 11.577 1.00 0.01 ATOM 860 CA MET A 99 27.114 3.392 11.817 1.00 0.02 ATOM 861 CB MET A 99 27.319 3.255 13.315 1.00 0.00 ATOM 862 CG MET A 99 28.783 3.315 13.704 1.00 0.01 ATOM 863 SD MET A 99 29.098 2.753 15.386 1.00 0.01 ATOM 864 CE MET A 99 28.443 1.074 15.249 1.00 0.02 ATOM 865 C MET A 99 27.989 2.367 11.103 1.00 0.01 ATOM 866 O MET A 99 28.949 2.735 10.412 1.00 0.01 ATOM 867 N LEU A 100 27.496 1.139 11.054 1.00 0.01 ATOM 869 CA LEU A 100 28.216 0.054 10.381 1.00 0.01 ATOM 870 CB LEU A 100 27.610 −1.265 10.847 1.00 0.01 ATOM 871 CG LEU A 100 28.374 −2.467 10.305 1.00 0.01 ATOM 872 CD1 LEU A 100 29.822 −2.450 10.784 1.00 0.01 ATOM 873 CD2 LEU A 100 27.691 −3.768 10.714 1.00 0.01 ATOM 874 C LEU A 100 28.104 0.162 8.862 1.00 0.01 ATOM 875 O LEU A 100 29.088 −0.102 8.160 1.00 0.01 ATOM 876 N SER A 101 27.047 0.812 8.397 1.00 0.01 ATOM 878 CA SER A 101 26.872 1.083 6.962 1.00 0.01 ATOM 879 CB SER A 101 25.395 1.301 6.668 1.00 0.00 ATOM 880 OG SER A 101 24.997 2.495 7.322 1.00 0.00 ATOM 881 C SER A 101 27.673 2.295 6.469 1.00 0.01 ATOM 882 O SER A 101 27.547 2.678 5.304 1.00 0.00 ATOM 883 N LEU A 102 28.450 2.909 7.350 1.00 0.01 ATOM 885 CA LEU A 102 29.438 3.915 6.944 1.00 0.01 ATOM 886 CB LEU A 102 29.500 5.012 7.994 1.00 0.00 ATOM 887 CG LEU A 102 28.356 5.997 7.809 1.00 0.01 ATOM 888 CD1 LEU A 102 28.419 7.100 8.855 1.00 0.01 ATOM 889 CD2 LEU A 102 28.408 6.601 6.411 1.00 0.01 ATOM 890 C LEU A 102 30.833 3.318 6.730 1.00 0.00 ATOM 891 O LEU A 102 31.800 4.067 6.540 1.00 0.01 ATOM 892 N GLY A 103 30.947 2.008 6.874 1.00 0.02 ATOM 894 CA GLY A 103 32.193 1.306 6.555 1.00 0.02 ATOM 895 C GLY A 103 31.876 0.155 5.611 1.00 0.01 ATOM 896 O GLY A 103 32.740 −0.334 4.866 1.00 0.02 ATOM 897 N ALA A 104 30.658 −0.337 5.756 1.00 0.01 ATOM 899 CA ALA A 104 30.107 −1.315 4.823 1.00 0.00 ATOM 900 CB ALA A 104 28.828 −1.898 5.410 1.00 0.60 ATOM 901 C ALA A 104 29.798 −0.648 3.494 1.00 0.01 ATOM 902 O ALA A 104 29.377 0.511 3.431 1.00 0.01 ATOM 903 N HIS A 105 30.084 −1.371 2.434 1.00 0.01 ATOM 905 CA HIS A 105 29.819 −0.868 1.093 1.00 0.01 ATOM 906 CB HIS A 105 31.163 −0.638 0.413 1.00 0.36 ATOM 907 CG HIS A 105 31.157 0.500 −0.582 1.00 1.35 ATOM 908 ND1 HIS A 105 30.544 1.685 −0.417 1.00 1.70 ATOM 910 CE1 HIS A 105 30.748 2.447 −1.511 1.00 2.51 ATOM 911 NE2 HIS A 105 31.505 1.730 −2.373 1.00 2.95 ATOM 912 CD2 HIS A 105 31.767 0.527 −1.813 1.00 2.35 ATOM 913 C HIS A 105 28.971 −1.875 0.322 1.00 0.01 ATOM 914 O HIS A 105 28.605 −2.934 0.858 1.00 0.01 ATOM 915 N SER A 106 28.645 −1.518 −0.912 1.00 0.01 ATOM 917 CA SER A 106 27.870 −2.369 −1.833 1.00 0.00 ATOM 918 CB SER A 106 28.789 −3.466 −2.358 1.00 0.30 ATOM 919 OG SER A 106 29.945 −2.846 −2.902 1.00 0.54 ATOM 920 C SER A 106 26.661 −3.026 −1.177 1.00 0.01 ATOM 921 O SER A 106 25.993 −2.427 −0.326 1.00 0.01 ATOM 922 N VAL A 107 26.547 −4.326 −1.403 1.00 0.01 ATOM 924 CA VAL A 107 25.393 −5.106 −0.926 1.00 0.01 ATOM 925 CB VAL A 107 25.402 −6.429 −1.689 1.00 0.37 ATOM 926 CG1 VAL A 107 24.244 −7.339 −1.287 1.00 0.53 ATOM 927 CG2 VAL A 107 25.369 −6.171 −3.191 1.00 0.37 ATOM 928 C VAL A 107 25.391 −5.361 0.589 1.00 0.01 ATOM 929 O VAL A 107 24.304 −5.492 1.162 1.00 0.01 ATOM 930 N THR A 108 26.506 −5.109 1.255 1.00 0.02 ATOM 932 CA THR A 108 26.545 −5.286 2.706 1.00 0.01 ATOM 933 CB THR A 108 28.003 −5.413 3.133 1.00 0.00 ATOM 934 OG1 THR A 108 28.524 −6.614 2.573 1.00 0.01 ATOM 935 CG2 THR A 108 28.144 −5.507 4.645 1.00 0.01 ATOM 936 C THR A 108 25.891 −4.079 3.374 1.00 0.01 ATOM 937 O THR A 108 24.945 −4.252 4.154 1.00 0.00 ATOM 938 N LYS A 109 26.119 −2.925 2.767 1.00 0.01 ATOM 940 CA LYS A 109 25.517 −1.675 3.238 1.00 0.01 ATOM 941 CB LYS A 109 26.307 −0.543 2.601 1.00 0.10 ATOM 942 CG LYS A 109 25.738 0.831 2.919 1.00 0.14 ATOM 943 CD LYS A 109 26.456 1.893 2.098 1.00 0.38 ATOM 944 CE LYS A 109 26.342 1.577 0.611 1.00 1.01 ATOM 945 NZ LYS A 109 27.041 2.585 −0.201 1.00 1.35 ATOM 946 C LYS A 109 24.061 −1.567 2.799 1.00 0.01 ATOM 947 O LYS A 109 23.203 −1.164 3.596 1.00 0.01 ATOM 948 N THR A 110 23.758 −2.201 1.678 1.00 0.01 ATOM 950 CA THR A 110 22.396 −2.187 1.145 1.00 0.00 ATOM 951 CB THR A 110 22.447 −2.650 −0.307 1.00 0.09 ATOM 952 OG1 THR A 110 23.256 −1.733 −1.031 1.00 0.10 ATOM 953 CG2 THR A 110 21.064 −2.670 −0.949 1.00 0.15 ATOM 954 C THR A 110 21.466 −3.093 1.945 1.00 0.01 ATOM 955 O THR A 110 20.337 −2.678 2.241 1.00 0.01 ATOM 956 N GLN A 111 22.002 −4.168 2.504 1.00 0.01 ATOM 958 CA GLN A 111 21.181 −5.033 3.350 1.00 0.00 ATOM 959 CB GLN A 111 21.872 −6.371 3.549 1.00 0.18 ATOM 960 CG GLN A 111 21.854 −7.232 2.297 1.00 0.37 ATOM 961 CD GLN A 111 22.526 −8.554 2.636 1.00 0.89 ATOM 962 OE1 GLN A 111 23.234 −8.651 3.648 1.00 1.44 ATOM 963 NE2 GLN A 111 22.246 −9.572 1.842 1.00 1.73 ATOM 966 C GLN A 111 20.952 −4.409 4.714 1.00 0.01 ATOM 967 O GLN A 111 19.821 −4.458 5.211 1.00 0.01 ATOM 968 N ILE A 112 21.915 −3.630 5.179 1.00 0.01 ATOM 970 CA ILE A 112 21.757 −2.939 6.459 1.00 0.00 ATOM 971 CB ILE A 112 23.092 −2.316 6.844 1.00 0.01 ATOM 972 CG2 ILE A 112 22.932 −1.466 8.094 1.00 0.01 ATOM 973 CG1 ILE A 112 24.156 −3.385 7.060 1.00 0.01 ATOM 974 CD1 ILE A 112 25.506 −2.767 7.404 1.00 0.01 ATOM 975 C ILE A 112 20.698 −1.846 6.371 1.00 0.00 ATOM 976 O ILE A 112 19.735 −1.868 7.152 1.00 0.01 ATOM 977 N LEU A 113 20.717 −1.103 5.277 1.00 0.01 ATOM 979 CA LEU A 113 19.765 −0.004 5.119 1.00 0.01 ATOM 980 CB LEU A 113 20.218 0.869 3.959 1.00 0.01 ATOM 981 CG LEU A 113 21.566 1.512 4.267 1.00 0.01 ATOM 982 CD1 LEU A 113 22.129 2.218 3.041 1.00 0.01 ATOM 983 CD2 LEU A 113 21.471 2.468 5.452 1.00 0.01 ATOM 984 C LEU A 113 18.348 −0.507 4.883 1.00 0.01 ATOM 985 O LEU A 113 17.476 −0.187 5.702 1.00 0.01 ATOM 986 N GLN A 114 18.193 −1.516 4.040 1.00 0.01 ATOM 988 CA GLN A 114 16.849 −2.042 3.757 1.00 0.01 ATOM 989 CB GLN A 114 16.906 −2.855 2.468 1.00 0.08 ATOM 990 CG GLN A 114 17.175 −1.980 1.243 1.00 0.98 ATOM 991 CD GLN A 114 15.896 −1.331 0.705 1.00 1.17 ATOM 992 OE1 GLN A 114 14.925 −1.088 1.431 1.00 0.48 ATOM 993 NE2 GLN A 114 15.902 −1.091 −0.596 1.00 2.23 ATOM 996 C GLN A 114 16.290 −2.901 4.897 1.00 0.01 ATOM 997 O GLN A 114 15.081 −2.841 5.146 1.00 0.01 ATOM 998 N GLY A 115 17.167 −3.418 5.744 1.00 0.01 ATOM 1000 CA GLY A 115 16.750 −4.161 6.941 1.00 0.01 ATOM 1001 C GLY A 115 16.228 −3.226 8.030 1.00 0.01 ATOM 1002 O GLY A 115 15.375 −3.613 8.837 1.00 0.01 ATOM 1003 N LEU A 116 16.724 −1.997 8.030 1.00 0.00 ATOM 1005 CA LEU A 116 16.217 −0.954 8.933 1.00 0.00 ATOM 1006 CB LEU A 116 17.330 0.055 9.176 1.00 0.01 ATOM 1007 CG LEU A 116 18.543 −0.556 9.859 1.00 0.01 ATOM 1008 CD1 LEU A 116 19.679 0.460 9.928 1.00 0.01 ATOM 1009 CD2 LEU A 116 18.183 −1.064 11.249 1.00 0.01 ATOM 1010 C LEU A 116 15.011 −0.197 8.360 1.00 0.00 ATOM 1011 O LEU A 116 14.511 0.734 9.004 1.00 0.00 ATOM 1012 N GLY A 117 14.620 −0.516 7.136 1.00 0.01 ATOM 1014 CA GLY A 117 13.451 0.115 6.517 1.00 0.00 ATOM 1015 C GLY A 117 13.831 1.183 5.497 1.00 0.00 ATOM 1016 O GLY A 117 12.957 1.814 4.890 1.00 0.01 ATOM 1017 N PHE A 118 15.121 1.368 5.286 1.00 0.00 ATOM 1019 CA PHE A 118 15.586 2.450 4.416 1.00 0.00 ATOM 1020 CB PHE A 118 16.933 2.952 4.908 1.00 0.01 ATOM 1021 CG PHE A 118 16.806 3.634 6.262 1.00 0.01 ATOM 1022 CD1 PHE A 118 16.182 4.872 6.344 1.00 0.00 ATOM 1023 CE1 PHE A 118 16.039 5.498 7.574 1.00 0.00 ATOM 1024 CZ PHE A 118 16.517 4.885 8.723 1.00 0.01 ATOM 1025 CE2 PHE A 118 17.143 3.648 8.640 1.00 0.01 ATOM 1026 CD2 PHE A 118 17.289 3.023 7.410 1.00 0.01 ATOM 1027 C PHE A 118 15.643 2.029 2.958 1.00 0.01 ATOM 1028 O PHE A 118 16.641 1.497 2.448 1.00 0.00 ATOM 1029 N ASN A 119 14.538 2.332 2.302 1.00 0.01 ATOM 1031 CA ASN A 119 14.351 2.050 0.884 1.00 0.00 ATOM 1032 CB ASN A 119 12.865 2.195 0.593 1.00 0.01 ATOM 1033 CG ASN A 119 12.593 1.896 −0.871 1.00 0.01 ATOM 1034 OD1 ASN A 119 12.727 2.781 −1.726 1.00 0.00 ATOM 1035 ND2 ASN A 119 12.292 0.642 −1.155 1.00 0.00 ATOM 1038 C ASN A 119 15.162 2.997 0.007 1.00 0.00 ATOM 1039 O ASN A 119 14.742 4.127 −0.281 1.00 0.01 ATOM 1040 N LEU A 120 16.152 2.409 −0.642 1.00 0.00 ATOM 1042 CA LEU A 120 17.113 3.144 −1.480 1.00 0.00 ATOM 1043 CB LEU A 120 18.373 2.294 −1.585 1.00 0.01 ATOM 1044 CG LEU A 120 18.987 2.053 −0.209 1.00 0.00 ATOM 1045 CD1 LEU A 120 20.142 1.062 −0.290 1.00 0.00 ATOM 1046 CD2 LEU A 120 19.439 3.363 0.430 1.00 0.01 ATOM 1047 C LEU A 120 16.621 3.502 −2.889 1.00 0.01 ATOM 1048 O LEU A 120 17.432 3.878 −3.742 1.00 0.01 ATOM 1049 N THR A 121 15.343 3.312 −3.176 1.00 0.01 ATOM 1051 CA THR A 121 14.820 3.787 −4.454 1.00 0.00 ATOM 1052 CB THR A 121 13.901 2.756 −5.098 1.00 0.00 ATOM 1053 OG1 THR A 121 12.623 2.809 −4.484 1.00 0.01 ATOM 1054 CG2 THR A 121 14.462 1.342 −4.991 1.00 0.01 ATOM 1055 C THR A 121 14.079 5.104 −4.233 1.00 0.01 ATOM 1056 O THR A 121 13.539 5.682 −5.182 1.00 0.00 ATOM 1057 N HIS A 122 13.997 5.525 −2.980 1.00 0.01 ATOM 1059 CA HIS A 122 13.428 6.835 −2.669 1.00 0.01 ATOM 1060 CB HIS A 122 12.089 6.647 −1.963 1.00 0.29 ATOM 1061 CG HIS A 122 11.014 5.994 −2.808 1.00 0.30 ATOM 1062 ND1 HIS A 122 10.628 4.704 −2.763 1.00 1.03 ATOM 1064 CE1 HIS A 122 9.642 4.505 −3.661 1.00 0.88 ATOM 1065 NE2 HIS A 122 9.411 5.682 −4.286 1.00 0.56 ATOM 1066 CD2 HIS A 122 10.249 6.610 −3.771 1.00 0.88 ATOM 1067 C HIS A 122 14.354 7.643 −1.765 1.00 0.00 ATOM 1068 O HIS A 122 14.502 8.859 −1.949 1.00 0.01 ATOM 1069 N THR A 123 14.987 6.970 −0.818 1.00 0.00 ATOM 1071 CA THR A 123 15.788 7.677 0.191 1.00 0.01 ATOM 1072 CB THR A 123 15.631 6.987 1.549 1.00 0.59 ATOM 1073 OG1 THR A 123 16.236 5.700 1.515 1.00 1.04 ATOM 1074 CG2 THR A 123 14.166 6.821 1.939 1.00 0.99 ATOM 1075 C THR A 123 17.270 7.773 −0.171 1.00 0.00 ATOM 1076 O THR A 123 17.948 6.768 −0.415 1.00 0.00 ATOM 1077 N PRO A 124 17.754 9.003 −0.197 1.00 0.01 ATOM 1078 CA PRO A 124 19.196 9.259 −0.178 1.00 0.00 ATOM 1079 CB PRO A 124 19.325 10.729 −0.438 1.00 0.01 ATOM 1080 CG PRO A 124 17.953 11.375 −0.308 1.00 0.01 ATOM 1081 CD PRO A 124 16.977 10.237 −0.053 1.00 0.01 ATOM 1082 C PRO A 124 19.803 8.894 1.177 1.00 0.01 ATOM 1083 O PRO A 124 19.255 9.238 2.234 1.00 0.01 ATOM 1084 N GLU A 125 21.035 8.412 1.136 1.00 0.01 ATOM 1086 CA GLU A 125 21.718 7.981 2.366 1.00 0.00 ATOM 1087 CB GLU A 125 22.887 7.071 2.009 1.00 0.00 ATOM 1088 CG GLU A 125 22.398 5.759 1.407 1.00 0.01 ATOM 1089 CD GLU A 125 23.581 4.845 1.106 1.00 0.01 ATOM 1090 OE1 GLU A 125 24.620 5.042 1.720 1.00 0.01 ATOM 1091 OE2 GLU A 125 23.455 4.043 0.191 1.00 0.01 ATOM 1092 C GLU A 125 22.215 9.141 3.231 1.00 0.01 ATOM 1093 O GLU A 125 22.304 8.970 4.451 1.00 0.01 ATOM 1094 N SER A 126 22.222 10.346 2.683 1.00 0.01 ATOM 1096 CA SER A 126 22.599 11.526 3.473 1.00 0.01 ATOM 1097 CB SER A 126 22.978 12.656 2.523 1.00 0.17 ATOM 1098 OG SER A 126 21.811 13.035 1.805 1.00 0.54 ATOM 1099 C SER A 126 21.460 11.996 4.384 1.00 0.01 ATOM 1100 O SER A 126 21.710 12.718 5.358 1.00 0.01 ATOM 1101 N ALA A 127 20.253 11.509 4.143 1.00 0.00 ATOM 1103 CA ALA A 127 19.143 11.802 5.044 1.00 0.01 ATOM 1104 CB ALA A 127 17.859 11.898 4.228 1.00 0.11 ATOM 1105 C ALA A 127 19.023 10.688 6.077 1.00 0.01 ATOM 1106 O ALA A 127 18.841 10.972 7.265 1.00 0.01 ATOM 1107 N ILE A 128 19.457 9.504 5.678 1.00 0.01 ATOM 1109 CA ILE A 128 19.417 8.335 6.561 1.00 0.01 ATOM 1110 CB ILE A 128 19.720 7.113 5.701 1.00 0.01 ATOM 1111 CG2 ILE A 128 19.813 5.854 6.554 1.00 0.01 ATOM 1112 CG1 ILE A 128 18.673 6.954 4.605 1.00 0.01 ATOM 1113 CD1 ILE A 128 18.998 5.776 3.693 1.00 0.01 ATOM 1114 C ILE A 128 20.457 8.442 7.670 1.00 0.00 ATOM 1115 O ILE A 128 20.121 8.299 8.854 1.00 0.01 ATOM 1116 N HIS A 129 21.625 8.948 7.311 1.00 0.00 ATOM 1118 CA HIS A 129 22.695 9.088 8.292 1.00 0.01 ATOM 1119 CB HIS A 129 24.020 9.140 7.544 1.00 0.01 ATOM 1120 CG HIS A 129 24.348 7.770 6.990 1.00 0.00 ATOM 1121 ND1 HIS A 129 24.406 6.646 7.719 1.00 0.01 ATOM 1123 CE1 HIS A 129 24.704 5.605 6.914 1.00 0.01 ATOM 1124 NE2 HIS A 129 24.842 6.088 5.659 1.00 0.01 ATOM 1125 CD2 HIS A 129 24.631 7.425 5.690 1.00 0.00 ATOM 1126 C HIS A 129 22.490 10.286 9.208 1.00 0.01 ATOM 1127 O HIS A 129 22.551 10.086 10.429 1.00 0.01 ATOM 1128 N GLN A 130 21.884 11.347 8.692 1.00 0.00 ATOM 1130 CA GLN A 130 21.584 12.495 9.554 1.00 0.01 ATOM 1131 CB GLN A 130 21.345 13.744 8.723 1.00 0.17 ATOM 1132 CG GLN A 130 22.630 14.305 8.135 1.00 0.98 ATOM 1133 CD GLN A 130 22.347 15.685 7.550 1.00 1.72 ATOM 1134 OE1 GLN A 130 22.530 16.708 8.220 1.00 2.37 ATOM 1135 NE2 GLN A 130 21.885 15.698 6.313 1.00 2.56 ATOM 1138 C GLN A 130 20.372 12.251 10.448 1.00 0.01 ATOM 1139 O GLN A 130 20.352 12.767 11.570 1.00 0.00 ATOM 1140 N GLY A 131 19.527 11.301 10.080 1.00 0.01 ATOM 1142 CA GLY A 131 18.427 10.871 10.943 1.00 0.01 ATOM 1143 C GLY A 131 18.960 10.364 12.275 1.00 0.01 ATOM 1144 O GLY A 131 18.723 10.998 13.315 1.00 0.01 ATOM 1145 N PHE A 132 19.895 9.429 12.203 1.00 0.01 ATOM 1147 CA PHE A 132 20.463 8.857 13.427 1.00 0.01 ATOM 1148 CB PHE A 132 21.059 7.497 13.110 1.00 0.01 ATOM 1149 CG PHE A 132 19.987 6.504 12.696 1.00 0.01 ATOM 1150 CD1 PHE A 132 18.916 6.253 13.543 1.00 0.01 ATOM 1151 CE1 PHE A 132 17.930 5.353 13.166 1.00 0.01 ATOM 1152 CZ PHE A 132 18.018 4.704 11.942 1.00 0.01 ATOM 1153 CE2 PHE A 132 19.088 4.957 11.095 1.00 0.01 ATOM 1154 CD2 PHE A 132 20.071 5.861 11.470 1.00 0.01 ATOM 1155 C PHE A 132 21.504 9.756 14.083 1.00 0.01 ATOM 1156 O PHE A 132 21.634 9.705 15.310 1.00 0.01 ATOM 1157 N GLN A 133 22.021 10.734 13.358 1.00 0.00 ATOM 1159 CA GLN A 133 22.907 11.715 13.988 1.00 0.01 ATOM 1160 CB GLN A 133 23.676 12.464 12.912 1.00 0.01 ATOM 1161 CG GLN A 133 24.601 11.545 12.125 1.00 0.01 ATOM 1162 CD GLN A 133 25.235 12.362 11.008 1.00 0.01 ATOM 1163 OE1 GLN A 133 25.151 12.017 9.820 1.00 0.00 ATOM 1164 NE2 GLN A 133 25.763 13.506 11.403 1.00 0.00 ATOM 1167 C GLN A 133 22.132 12.726 14.832 1.00 0.01 ATOM 1168 O GLN A 133 22.580 13.036 15.942 1.00 0.01 ATOM 1169 N HIS A 134 20.896 13.025 14.459 1.00 0.01 ATOM 1171 CA HIS A 134 20.097 13.966 15.263 1.00 0.01 ATOM 1172 CB HIS A 134 18.982 14.624 14.460 1.00 0.18 ATOM 1173 CG HIS A 134 19.312 15.245 13.125 1.00 0.86 ATOM 1174 ND1 HIS A 134 18.393 15.644 12.235 1.00 1.74 ATOM 1176 CE1 HIS A 134 19.018 16.128 11.144 1.00 2.27 ATOM 1177 NE2 HIS A 134 20.344 16.050 11.364 1.00 2.14 ATOM 1178 CD2 HIS A 134 20.545 15.536 12.597 1.00 1.59 ATOM 1179 C HIS A 134 19.412 13.225 16.393 1.00 0.01 ATOM 1180 O HIS A 134 19.141 13.801 17.454 1.00 0.01 ATOM 1181 N LEU A 135 19.310 11.920 16.226 1.00 0.01 ATOM 1183 CA LEU A 135 18.802 11.066 17.292 1.00 0.01 ATOM 1184 CB LEU A 135 18.447 9.732 16.655 1.00 0.01 ATOM 1185 CG LEU A 135 17.628 8.844 17.574 1.00 0.01 ATOM 1186 CD1 LEU A 135 16.426 9.600 18.133 1.00 0.01 ATOM 1187 CD2 LEU A 135 17.182 7.601 16.813 1.00 0.01 ATOM 1188 C LEU A 135 19.875 10.912 18.369 1.00 0.01 ATOM 1189 O LEU A 135 19.565 11.061 19.557 1.00 0.01 ATOM 1190 N VAL A 136 21.129 10.952 17.944 1.00 0.01 ATOM 1192 CA VAL A 136 22.252 11.007 18.884 1.00 0.01 ATOM 1193 CB VAL A 136 23.538 10.706 18.115 1.00 0.27 ATOM 1194 CG1 VAL A 136 24.779 11.158 18.874 1.00 0.79 ATOM 1195 CG2 VAL A 136 23.631 9.230 17.746 1.00 0.30 ATOM 1196 C VAL A 136 22.357 12.377 19.558 1.00 0.00 ATOM 1197 O VAL A 136 22.491 12.418 20.786 1.00 0.01 ATOM 1198 N HIS A 137 22.011 13.439 18.844 1.00 0.01 ATOM 1200 CA HIS A 137 22.037 14.787 19.436 1.00 0.02 ATOM 1201 CB HIS A 137 21.806 15.838 18.348 1.00 0.16 ATOM 1202 CG HIS A 137 22.837 15.906 17.234 1.00 0.20 ATOM 1203 ND1 HIS A 137 22.648 16.421 16.003 1.00 0.23 ATOM 1205 CE1 HIS A 137 23.784 16.303 15.288 1.00 0.21 ATOM 1206 NE2 HIS A 137 24.709 15.722 16.085 1.00 0.23 ATOM 1207 CD2 HIS A 137 24.145 15.483 17.290 1.00 0.25 ATOM 1208 C HIS A 137 20.950 14.950 20.499 1.00 0.01 ATOM 1209 O HIS A 137 21.257 15.358 21.627 1.00 0.01 ATOM 1210 N SER A 138 19.784 14.376 20.239 1.00 0.01 ATOM 1212 CA SER A 138 18.665 14.459 21.191 1.00 0.01 ATOM 1213 CB SER A 138 17.355 14.205 20.449 1.00 0.30 ATOM 1214 OG SER A 138 17.373 12.882 19.930 1.00 0.53 ATOM 1215 C SER A 138 18.799 13.466 22.348 1.00 0.01 ATOM 1216 O SER A 138 18.125 13.616 23.375 1.00 0.00 ATOM 1217 N LEU A 139 19.717 12.523 22.225 1.00 0.01 ATOM 1219 CA LEU A 139 20.031 11.612 23.325 1.00 0.01 ATOM 1220 CB LEU A 139 20.118 10.188 22.797 1.00 0.01 ATOM 1221 CG LEU A 139 18.756 9.706 22.308 1.00 0.00 ATOM 1222 CD1 LEU A 139 18.857 8.310 21.708 1.00 0.01 ATOM 1223 CD2 LEU A 139 17.723 9.741 23.431 1.00 0.01 ATOM 1224 C LEU A 139 21.327 11.991 24.045 1.00 0.01 ATOM 1225 O LEU A 139 21.830 11.202 24.853 1.00 0.01 ATOM 1226 N THR A 140 21.881 13.152 23.737 1.00 0.01 ATOM 1228 CA THR A 140 23.071 13.626 24.453 1.00 0.01 ATOM 1229 CB THR A 140 24.276 13.631 23.517 1.00 0.52 ATOM 1230 OG1 THR A 140 23.934 14.391 22.365 1.00 1.05 ATOM 1231 CG2 THR A 140 24.672 12.227 23.071 1.00 0.85 ATOM 1232 C THR A 140 22.886 15.029 25.026 1.00 0.01 ATOM 1233 O THR A 140 23.881 15.742 25.199 1.00 0.01 ATOM 1234 N VAL A 141 21.650 15.419 25.304 1.00 0.02 ATOM 1236 CA VAL A 141 21.356 16.781 25.793 1.00 0.01 ATOM 1237 CB VAL A 141 19.842 16.978 25.741 1.00 0.79 ATOM 1238 CG1 VAL A 141 19.440 18.380 26.189 1.00 1.02 ATOM 1239 CG2 VAL A 141 19.312 16.707 24.338 1.00 0.94 ATOM 1240 C VAL A 141 21.861 17.027 27.220 1.00 0.01 ATOM 1241 O VAL A 141 21.240 16.603 28.202 1.00 0.01 ATOM 1242 N PRO A 142 22.865 17.887 27.325 1.00 0.01 ATOM 1243 CA PRO A 142 23.559 18.090 28.603 1.00 0.02 ATOM 1244 CB PRO A 142 24.852 18.745 28.224 1.00 0.01 ATOM 1245 CG PRO A 142 24.793 19.171 26.763 1.00 0.01 ATOM 1246 CD PRO A 142 23.476 18.639 26.223 1.00 0.02 ATOM 1247 C PRO A 142 22.794 18.975 29.594 1.00 0.01 ATOM 1248 O PRO A 142 22.973 18.832 30.808 1.00 0.02 ATOM 1249 N SER A 143 21.846 19.756 29.097 1.00 0.01 ATOM 1251 CA SER A 143 21.070 20.655 29.962 1.00 0.02 ATOM 1252 CB SER A 143 20.710 21.911 29.179 1.00 1.07 ATOM 1253 OG SER A 143 19.787 21.539 28.164 1.00 1.15 ATOM 1254 C SER A 143 19.786 19.997 30.459 1.00 0.01 ATOM 1255 O SER A 143 19.067 20.572 31.283 1.00 0.02 ATOM 1256 N LYS A 144 19.510 18.806 29.956 1.00 0.01 ATOM 1258 CA LYS A 144 18.340 18.042 30.382 1.00 0.02 ATOM 1259 CB LYS A 144 17.101 18.505 29.620 1.00 0.39 ATOM 1260 CG LYS A 144 15.850 17.808 30.147 1.00 0.73 ATOM 1261 CD LYS A 144 15.693 18.028 31.649 1.00 0.88 ATOM 1262 CE LYS A 144 14.516 17.238 32.208 1.00 1.90 ATOM 1263 NZ LYS A 144 14.372 17.446 33.657 1.00 2.56 ATOM 1264 C LYS A 144 18.625 16.575 30.111 1.00 0.01 ATOM 1265 O LYS A 144 18.213 16.009 29.089 1.00 0.02 ATOM 1266 N ASP A 145 19.330 15.963 31.045 1.00 0.01 ATOM 1268 CA ASP A 145 19.767 14.583 30.849 1.00 0.00 ATOM 1269 CB ASP A 145 21.057 14.363 31.629 1.00 0.63 ATOM 1270 CG ASP A 145 21.709 13.068 31.158 1.00 1.02 ATOM 1271 OD1 ASP A 145 22.537 12.543 31.886 1.00 1.80 ATOM 1272 OD2 ASP A 145 21.406 12.669 30.041 1.00 0.62 ATOM 1273 C ASP A 145 18.703 13.568 31.272 1.00 0.01 ATOM 1274 O ASP A 145 18.729 13.027 32.381 1.00 0.01 ATOM 1275 N LEU A 146 17.768 13.333 30.364 1.00 0.01 ATOM 1277 CA LEU A 146 16.796 12.240 30.509 1.00 0.02 ATOM 1278 CB LEU A 146 15.424 12.731 30.037 1.00 0.01 ATOM 1279 CG LEU A 146 14.351 11.649 30.160 1.00 0.01 ATOM 1280 CD1 LEU A 146 14.209 11.160 31.599 1.00 0.01 ATOM 1281 CD2 LEU A 146 13.007 12.109 29.612 1.00 0.01 ATOM 1282 C LEU A 146 17.253 11.058 29.650 1.00 0.02 ATOM 1283 O LEU A 146 16.750 9.934 29.763 1.00 0.01 ATOM 1284 N THR A 147 18.323 11.305 28.915 1.00 0.00 ATOM 1286 CA THR A 147 18.804 10.382 27.894 1.00 0.01 ATOM 1287 CB THR A 147 19.914 11.089 27.153 1.00 2.66 ATOM 1288 OG1 THR A 147 21.122 10.931 27.882 1.00 3.01 ATOM 1289 CG2 THR A 147 19.599 12.574 27.002 1.00 3.30 ATOM 1290 C THR A 147 19.313 9.064 28.460 1.00 0.01 ATOM 1291 O THR A 147 19.523 8.894 29.666 1.00 0.01 ATOM 1292 N LEU A 148 19.478 8.131 27.543 1.00 0.01 ATOM 1294 CA LEU A 148 19.785 6.740 27.901 1.00 0.00 ATOM 1295 CB LEU A 148 19.422 5.797 26.747 1.00 0.01 ATOM 1296 CG LEU A 148 17.933 5.672 26.394 1.00 0.01 ATOM 1297 CD1 LEU A 148 17.000 6.009 27.550 1.00 0.00 ATOM 1298 CD2 LEU A 148 17.554 6.469 25.149 1.00 0.01 ATOM 1299 C LEU A 148 21.263 6.540 28.222 1.00 0.01 ATOM 1300 O LEU A 148 22.140 7.211 27.660 1.00 0.02 ATOM 1301 N LYS A 149 21.526 5.577 29.090 1.00 0.01 ATOM 1303 CA LYS A 149 22.899 5.138 29.373 1.00 0.01 ATOM 1304 CB LYS A 149 22.989 4.568 30.784 1.00 0.59 ATOM 1305 CG LYS A 149 22.743 5.637 31.840 1.00 0.67 ATOM 1306 CD LYS A 149 22.935 5.064 33.239 1.00 0.79 ATOM 1307 CE LYS A 149 24.323 4.451 33.397 1.00 0.79 ATOM 1308 NZ LYS A 149 24.486 3.849 34.730 1.00 1.03 ATOM 1309 C LYS A 149 23.310 4.070 28.367 1.00 0.01 ATOM 1310 O LYS A 149 23.169 2.862 28.605 1.00 0.01 ATOM 1311 N MET A 150 23.712 4.541 27.201 1.00 0.01 ATOM 1313 CA MET A 150 24.103 3.652 26.110 1.00 0.02 ATOM 1314 CB MET A 150 23.980 4.407 24.794 1.00 0.87 ATOM 1315 CG MET A 150 22.548 4.877 24.562 1.00 1.11 ATOM 1316 SD MET A 150 22.254 5.732 22.997 1.00 2.15 ATOM 1317 CE MET A 150 22.722 4.406 21.860 1.00 2.30 ATOM 1318 C MET A 150 25.525 3.136 26.285 1.00 0.01 ATOM 1319 O MET A 150 26.345 3.715 27.004 1.00 0.02 ATOM 1320 N GLY A 151 25.762 1.986 25.684 1.00 0.01 ATOM 1322 CA GLY A 151 27.084 1.360 25.688 1.00 0.02 ATOM 1323 C GLY A 151 27.333 0.658 24.358 1.00 0.01 ATOM 1324 O GLY A 151 27.128 −0.558 24.235 1.00 0.01 ATOM 1325 N SER A 152 27.756 1.424 23.366 1.00 0.02 ATOM 1327 CA SER A 152 27.965 0.840 22.037 1.00 0.01 ATOM 1328 CB SER A 152 27.031 1.518 21.042 1.00 0.59 ATOM 1329 OG SER A 152 26.747 0.584 20.003 1.00 1.14 ATOM 1330 C SER A 152 29.430 0.954 21.604 1.00 0.01 ATOM 1331 O SER A 152 29.998 2.051 21.483 1.00 0.01 ATOM 1332 N ALA A 153 30.002 −0.201 21.312 1.00 0.01 ATOM 1334 CA ALA A 153 31.434 −0.285 21.014 1.00 0.01 ATOM 1335 CB ALA A 153 32.184 −0.564 22.308 1.00 0.16 ATOM 1336 C ALA A 153 31.785 −1.353 19.982 1.00 0.01 ATOM 1337 O ALA A 153 31.344 −2.509 20.054 1.00 0.01 ATOM 1338 N LEU A 154 32.661 −0.952 19.079 1.00 0.01 ATOM 1340 CA LEU A 154 33.177 −1.833 18.032 1.00 0.02 ATOM 1341 CB LEU A 154 33.318 −1.043 16.738 1.00 0.02 ATOM 1342 CG LEU A 154 31.967 −0.608 16.192 1.00 0.02 ATOM 1343 CD1 LEU A 154 32.149 0.287 14.974 1.00 0.02 ATOM 1344 CD2 LEU A 154 31.112 −1.821 15.843 1.00 0.01 ATOM 1345 C LEU A 154 34.545 −2.373 18.419 1.00 0.02 ATOM 1346 O LEU A 154 35.547 −1.646 18.423 1.00 0.02 ATOM 1347 N PHE A 155 34.580 −3.660 18.697 1.00 0.01 ATOM 1349 CA PHE A 155 35.833 −4.328 19.045 1.00 0.01 ATOM 1350 CB PHE A 155 35.588 −5.319 20.177 1.00 0.02 ATOM 1351 CG PHE A 155 35.233 −4.669 21.513 1.00 0.02 ATOM 1352 CD1 PHE A 155 33.905 −4.509 21.891 1.00 0.01 ATOM 1353 CE1 PHE A 155 33.595 −3.915 23.108 1.00 0.02 ATOM 1354 CZ PHE A 155 34.612 −3.487 23.951 1.00 0.02 ATOM 1355 CE2 PHE A 155 35.938 −3.656 23.579 1.00 0.02 ATOM 1356 CD2 PHE A 155 36.248 −4.248 22.362 1.00 0.01 ATOM 1357 C PHE A 155 36.396 −5.020 17.811 1.00 0.01 ATOM 1358 O PHE A 155 36.004 −6.140 17.456 1.00 0.01 ATOM 1359 N VAL A 156 37.292 −4.305 17.155 1.00 0.01 ATOM 1361 CA VAL A 156 37.900 −4.765 15.907 1.00 0.02 ATOM 1362 CB VAL A 156 38.348 −3.545 15.106 1.00 0.49 ATOM 1363 CG1 VAL A 156 37.177 −2.605 14.855 1.00 0.60 ATOM 1364 CG2 VAL A 156 39.465 −2.791 15.815 1.00 0.71 ATOM 1365 C VAL A 156 39.096 −5.655 16.208 1.00 0.02 ATOM 1366 O VAL A 156 39.663 −5.590 17.305 1.00 0.01 ATOM 1367 N LYS A 157 39.400 −6.547 15.284 1.00 0.01 ATOM 1369 CA LYS A 157 40.561 −7.422 15.450 1.00 0.01 ATOM 1370 CB LYS A 157 40.556 −8.448 14.320 1.00 0.81 ATOM 1371 CG LYS A 157 41.622 −9.520 14.506 1.00 0.87 ATOM 1372 CD LYS A 157 41.619 −10.513 13.352 1.00 1.04 ATOM 1373 CE LYS A 157 42.711 −11.559 13.538 1.00 0.96 ATOM 1374 NZ LYS A 157 44.032 −10.918 13.638 1.00 2.10 ATOM 1375 C LYS A 157 41.862 −6.621 15.450 1.00 0.02 ATOM 1376 O LYS A 157 42.037 −5.658 14.691 1.00 0.02 ATOM 1377 N LYS A 158 42.687 −6.932 16.434 1.00 0.01 ATOM 1379 CA LYS A 158 44.031 −6.371 16.530 1.00 0.01 ATOM 1380 CB LYS A 158 44.706 −7.030 17.731 1.00 0.36 ATOM 1381 CG LYS A 158 46.081 −6.452 18.039 1.00 1.34 ATOM 1382 CD LYS A 158 46.724 −7.176 19.214 1.00 1.37 ATOM 1383 CE LYS A 158 48.116 −6.632 19.509 1.00 2.23 ATOM 1384 NZ LYS A 158 48.728 −7.342 20.642 1.00 2.98 ATOM 1385 C LYS A 158 44.818 −6.663 15.256 1.00 0.02 ATOM 1386 O LYS A 158 44.750 −7.769 14.708 1.00 0.02 ATOM 1387 N GLU A 159 45.458 −5.616 14.752 1.00 0.02 ATOM 1389 CA GLU A 159 46.337 −5.662 13.568 1.00 0.00 ATOM 1390 CB GLU A 159 47.312 −6.837 13.652 1.00 0.97 ATOM 1391 CG GLU A 159 48.235 −6.729 14.860 1.00 1.47 ATOM 1392 CD GLU A 159 49.069 −7.997 14.999 1.00 1.57 ATOM 1393 OE1 GLU A 159 49.297 −8.396 16.135 1.00 2.13 ATOM 1394 OE2 GLU A 159 49.515 −8.508 13.982 1.00 1.77 ATOM 1395 C GLU A 159 45.561 −5.739 12.255 1.00 0.01 ATOM 1396 O GLU A 159 45.933 −6.505 11.360 1.00 0.02 ATOM 1397 N LEU A 160 44.478 −4.986 12.163 1.00 0.02 ATOM 1399 CA LEU A 160 43.803 −4.808 10.872 1.00 0.02 ATOM 1400 CB LEU A 160 42.291 −4.800 11.054 1.00 0.02 ATOM 1401 CG LEU A 160 41.736 −6.146 11.496 1.00 0.01 ATOM 1402 CD1 LEU A 160 40.224 −6.046 11.653 1.00 0.02 ATOM 1403 CD2 LEU A 160 42.095 −7.249 10.506 1.00 0.00 ATOM 1404 C LEU A 160 44.218 −3.479 10.256 1.00 0.00 ATOM 1405 O LEU A 160 44.754 −2.605 10.950 1.00 0.02 ATOM 1406 N GLN A 161 43.941 −3.317 8.974 1.00 0.01 ATOM 1408 CA GLN A 161 44.189 −2.029 8.312 1.00 0.01 ATOM 1409 CB GLN A 161 44.562 −2.265 6.857 1.00 0.92 ATOM 1410 CG GLN A 161 46.073 −2.179 6.681 1.00 1.52 ATOM 1411 CD GLN A 161 46.526 −0.743 6.938 1.00 1.99 ATOM 1412 OE1 GLN A 161 45.895 0.209 6.469 1.00 2.12 ATOM 1413 NE2 GLN A 161 47.601 −0.601 7.694 1.00 2.59 ATOM 1416 C GLN A 161 42.978 −1.108 8.421 1.00 0.00 ATOM 1417 O GLN A 161 42.227 −0.902 7.459 1.00 0.00 ATOM 1418 N LEU A 162 42.846 −0.533 9.605 1.00 0.02 ATOM 1420 CA LEU A 162 41.713 0.327 9.948 1.00 0.00 ATOM 1421 CB LEU A 162 41.655 0.461 11.464 1.00 0.02 ATOM 1422 CG LEU A 162 41.484 −0.895 12.139 1.00 0.02 ATOM 1423 CD1 LEU A 162 41.692 −0.783 13.644 1.00 0.01 ATOM 1424 CD2 LEU A 162 40.125 −1.510 11.816 1.00 0.02 ATOM 1425 C LEU A 162 41.847 1.709 9.324 1.00 0.00 ATOM 1426 O LEU A 162 42.798 2.456 9.587 1.00 0.02 ATOM 1427 N GLN A 163 40.861 2.041 8.516 1.00 0.01 ATOM 1429 CA GLN A 163 40.834 3.337 7.847 1.00 0.00 ATOM 1430 CB GLN A 163 39.974 3.191 6.604 1.00 0.67 ATOM 1431 CG GLN A 163 40.562 2.109 5.706 1.00 1.20 ATOM 1432 CD GLN A 163 39.575 1.724 4.613 1.00 2.06 ATOM 1433 OE1 GLN A 163 39.212 0.548 4.484 1.00 2.42 ATOM 1434 NE2 GLN A 163 39.127 2.710 3.859 1.00 2.92 ATOM 1437 C GLN A 163 40.291 4.420 8.768 1.00 0.01 ATOM 1438 O GLN A 163 39.235 4.271 9.398 1.00 0.02 ATOM 1439 N ALA A 164 40.912 5.584 8.666 1.00 0.00 ATOM 1441 CA ALA A 164 40.567 6.716 9.535 1.00 0.02 ATOM 1442 CB ALA A 164 41.691 7.742 9.468 1.00 0.37 ATOM 1443 C ALA A 164 39.244 7.384 9.171 1.00 0.01 ATOM 1444 O ALA A 164 38.627 8.022 10.030 1.00 0.00 ATOM 1445 N ASN A 165 38.696 7.036 8.019 1.00 0.02 ATOM 1447 CA ASN A 165 37.393 7.564 7.624 1.00 0.02 ATOM 1448 CB ASN A 165 37.238 7.379 6.122 1.00 0.32 ATOM 1449 CG ASN A 165 38.369 8.106 5.408 1.00 1.12 ATOM 1450 OD1 ASN A 165 39.322 7.482 4.925 1.00 1.77 ATOM 1451 ND2 ASN A 165 38.285 9.425 5.425 1.00 2.08 ATOM 1454 C ASN A 165 36.271 6.831 8.344 1.00 0.01 ATOM 1455 O ASN A 165 35.347 7.484 8.842 1.00 0.02 ATOM 1456 N PHE A 166 36.494 5.563 8.654 1.00 0.02 ATOM 1458 CA PHE A 166 35.481 4.817 9.395 1.00 0.01 ATOM 1459 CB PHE A 166 35.696 3.326 9.201 1.00 0.02 ATOM 1460 CG PHE A 166 34.703 2.464 9.972 1.00 0.02 ATOM 1461 CD1 PHE A 166 33.360 2.476 9.623 1.00 0.01 ATOM 1462 CE1 PHE A 166 32.453 1.687 10.317 1.00 0.01 ATOM 1463 CZ PHE A 166 32.889 0.892 11.369 1.00 0.02 ATOM 1464 CE2 PHE A 166 34.230 0.892 11.731 1.00 0.01 ATOM 1465 CD2 PHE A 166 35.137 1.681 11.035 1.00 0.01 ATOM 1466 C PHE A 166 35.573 5.138 10.874 1.00 0.01 ATOM 1467 O PHE A 166 34.534 5.303 11.524 1.00 0.02 ATOM 1468 N LEU A 167 36.769 5.495 11.315 1.00 0.01 ATOM 1470 CA LEU A 167 36.960 5.888 12.712 1.00 0.02 ATOM 1471 CB LEU A 167 38.457 5.931 13.000 1.00 0.02 ATOM 1472 CG LEU A 167 39.122 4.585 12.726 1.00 0.02 ATOM 1473 CD1 LEU A 167 40.639 4.684 12.848 1.00 0.01 ATOM 1474 CD2 LEU A 167 38.580 3.494 13.643 1.00 0.02 ATOM 1475 C LEU A 167 36.339 7.261 12.953 1.00 0.02 ATOM 1476 O LEU A 167 35.587 7.429 13.923 1.00 0.01 ATOM 1477 N GLY A 168 36.441 8.120 11.951 1.00 0.01 ATOM 1479 CA GLY A 168 35.776 9.427 11.965 1.00 0.01 ATOM 1480 C GLY A 168 34.261 9.276 12.052 1.00 0.01 ATOM 1481 O GLY A 168 33.673 9.677 13.063 1.00 0.01 ATOM 1482 N ASN A 169 33.691 8.508 11.136 1.00 0.02 ATOM 1484 CA ASN A 169 32.238 8.294 11.107 1.00 0.01 ATOM 1485 CB ASN A 169 31.922 7.334 9.965 1.00 0.23 ATOM 1486 CG ASN A 169 32.243 7.956 8.607 1.00 0.32 ATOM 1487 OD1 ASN A 169 32.241 9.183 8.452 1.00 1.33 ATOM 1488 ND2 ASN A 169 32.425 7.101 7.614 1.00 0.79 ATOM 1491 C ASN A 169 31.692 7.704 12.407 1.00 0.01 ATOM 1492 O ASN A 169 30.833 8.331 13.043 1.00 0.01 ATOM 1493 N VAL A 170 32.369 6.699 12.934 1.00 0.01 ATOM 1495 CA VAL A 170 31.893 6.039 14.149 1.00 0.01 ATOM 1496 CB VAL A 170 32.705 4.765 14.329 1.00 0.02 ATOM 1497 CG1 VAL A 170 32.420 4.094 15.663 1.00 0.01 ATOM 1498 CG2 VAL A 170 32.422 3.803 13.189 1.00 0.02 ATOM 1499 C VAL A 170 32.007 6.909 15.397 1.00 0.01 ATOM 1500 O VAL A 170 30.975 7.179 16.027 1.00 0.02 ATOM 1501 N LYS A 171 33.147 7.541 15.608 1.00 0.01 ATOM 1503 CA LYS A 171 33.345 8.264 16.867 1.00 0.01 ATOM 1504 CB LYS A 171 34.843 8.333 17.125 1.00 0.00 ATOM 1505 CG LYS A 171 35.458 6.940 17.120 1.00 0.02 ATOM 1506 CD LYS A 171 36.977 7.001 17.204 1.00 0.02 ATOM 1507 CE LYS A 171 37.580 5.606 17.105 1.00 0.02 ATOM 1508 NZ LYS A 171 39.050 5.655 17.129 1.00 0.02 ATOM 1509 C LYS A 171 32.773 9.677 16.841 1.00 0.01 ATOM 1510 O LYS A 171 32.098 10.082 17.793 1.00 0.01 ATOM 1511 N ARG A 172 32.835 10.316 15.688 1.00 0.01 ATOM 1513 CA ARG A 172 32.435 11.720 15.585 1.00 0.00 ATOM 1514 CB ARG A 172 33.397 12.362 14.591 1.00 0.23 ATOM 1515 CG ARG A 172 32.990 13.760 14.149 1.00 1.13 ATOM 1516 CD ARG A 172 33.933 14.237 13.052 1.00 1.10 ATOM 1517 NE ARG A 172 34.064 13.196 12.018 1.00 1.48 ATOM 1518 CZ ARG A 172 33.587 13.311 10.777 1.00 2.53 ATOM 1519 NH1 ARG A 172 33.721 12.299 9.917 1.00 3.39 ATOM 1520 NH2 ARG A 172 32.953 14.425 10.404 1.00 3.21 ATOM 1521 C ARG A 172 30.992 11.912 15.124 1.00 0.01 ATOM 1522 O ARG A 172 30.331 12.858 15.567 1.00 0.01 ATOM 1523 N LEU A 173 30.467 10.983 14.344 1.00 0.01 ATOM 1525 CA LEU A 173 29.098 11.161 13.854 1.00 0.01 ATOM 1526 CB LEU A 173 29.062 10.874 12.356 1.00 0.01 ATOM 1527 CG LEU A 173 29.977 11.809 11.572 1.00 0.02 ATOM 1528 CD1 LEU A 173 30.030 11.409 10.102 1.00 0.01 ATOM 1529 CD2 LEU A 173 29.540 13.263 11.717 1.00 0.01 ATOM 1530 C LEU A 173 28.103 10.255 14.571 1.00 0.01 ATOM 1531 O LEU A 173 26.901 10.546 14.576 1.00 0.02 ATOM 1532 N TYR A 174 28.592 9.176 15.160 1.00 0.01 ATOM 1534 CA TYR A 174 27.700 8.255 15.881 1.00 0.01 ATOM 1535 CB TYR A 174 27.665 6.919 15.148 1.00 0.01 ATOM 1536 CG TYR A 174 26.771 6.904 13.912 1.00 0.01 ATOM 1537 CD1 TYR A 174 27.282 7.217 12.659 1.00 0.01 ATOM 1538 CE1 TYR A 174 26.452 7.192 11.547 1.00 0.00 ATOM 1539 CZ TYR A 174 25.114 6.850 11.688 1.00 0.01 ATOM 1540 OH TYR A 174 24.338 6.654 10.568 1.00 0.01 ATOM 1541 CE2 TYR A 174 24.599 6.548 12.940 1.00 0.01 ATOM 1542 CD2 TYR A 174 25.429 6.576 14.052 1.00 0.01 ATOM 1543 C TYR A 174 28.088 8.028 17.342 1.00 0.01 ATOM 1544 O TYR A 174 27.429 7.233 18.025 1.00 0.01 ATOM 1545 N GLU A 175 29.085 8.764 17.816 1.00 0.02 ATOM 1547 CA GLU A 175 29.666 8.641 19.175 1.00 0.01 ATOM 1548 CB GLU A 175 28.893 9.507 20.187 1.00 2.11 ATOM 1549 CG GLU A 175 27.387 9.247 20.319 1.00 2.81 ATOM 1550 CD GLU A 175 27.038 8.241 21.419 1.00 3.91 ATOM 1551 OE1 GLU A 175 27.508 8.466 22.527 1.00 4.60 ATOM 1552 OE2 GLU A 175 26.072 7.518 21.217 1.00 4.31 ATOM 1553 C GLU A 175 29.848 7.202 19.666 1.00 0.02 ATOM 1554 O GLU A 175 29.416 6.852 20.768 1.00 0.02 ATOM 1555 N ALA A 176 30.497 6.378 18.863 1.00 0.01 ATOM 1557 CA ALA A 176 30.770 5.009 19.298 1.00 0.01 ATOM 1558 CB ALA A 176 30.085 4.021 18.371 1.00 0.31 ATOM 1559 C ALA A 176 32.265 4.741 19.371 1.00 0.02 ATOM 1560 O ALA A 176 33.063 5.261 18.581 1.00 0.01 ATOM 1561 N GLU A 177 32.630 3.913 20.331 1.00 0.01 ATOM 1563 CA GLU A 177 34.048 3.668 20.610 1.00 0.02 ATOM 1564 CB GLU A 177 34.154 3.396 22.101 1.00 0.01 ATOM 1565 CG GLU A 177 33.567 4.556 22.894 1.00 0.02 ATOM 1566 CD GLU A 177 33.460 4.182 24.366 1.00 0.01 ATOM 1567 OE1 GLU A 177 34.417 4.421 25.089 1.00 0.02 ATOM 1568 OE2 GLU A 177 32.414 3.674 24.741 1.00 0.02 ATOM 1569 C GLU A 177 34.603 2.482 19.829 1.00 0.02 ATOM 1570 O GLU A 177 34.006 1.401 19.833 1.00 0.02 ATOM 1571 N VAL A 178 35.710 2.696 19.138 1.00 0.01 ATOM 1573 CA VAL A 178 36.389 1.592 18.442 1.00 0.02 ATOM 1574 CB VAL A 178 36.763 2.034 17.029 1.00 0.21 ATOM 1575 CG1 VAL A 178 37.555 0.962 16.287 1.00 0.34 ATOM 1576 CG2 VAL A 178 35.525 2.417 16.230 1.00 0.34 ATOM 1577 C VAL A 178 37.641 1.164 19.208 1.00 0.02 ATOM 1578 O VAL A 178 38.536 1.975 19.474 1.00 0.01 ATOM 1579 N PHE A 179 37.673 −0.103 19.582 1.00 0.02 ATOM 1581 CA PHE A 179 38.813 −0.652 20.325 1.00 0.02 ATOM 1582 CB PHE A 179 38.331 −1.131 21.688 1.00 0.01 ATOM 1583 CG PHE A 179 37.785 −0.030 22.590 1.00 0.02 ATOM 1584 CD1 PHE A 179 38.610 1.013 22.989 1.00 0.01 ATOM 1585 CE1 PHE A 179 38.114 2.014 23.814 1.00 0.02 ATOM 1586 CZ PHE A 179 36.794 1.969 24.241 1.00 0.01 ATOM 1587 CE2 PHE A 179 35.969 0.926 23.841 1.00 0.01 ATOM 1588 CD2 PHE A 179 36.464 −0.074 23.015 1.00 0.02 ATOM 1589 C PHE A 179 39.456 −1.824 19.594 1.00 0.01 ATOM 1590 O PHE A 179 38.765 −2.745 19.145 1.00 0.02 ATOM 1591 N SER A 180 40.772 −1.770 19.469 1.00 0.02 ATOM 1593 CA SER A 180 41.520 −2.893 18.890 1.00 0.01 ATOM 1594 CB SER A 180 42.876 −2.402 18.405 1.00 0.08 ATOM 1595 OG SER A 180 43.587 −3.537 17.933 1.00 0.09 ATOM 1596 C SER A 180 41.713 −3.982 19.938 1.00 0.01 ATOM 1597 O SER A 180 42.463 −3.816 20.906 1.00 0.02 ATOM 1598 N THR A 181 41.050 −5.100 19.718 1.00 0.01 ATOM 1600 CA THR A 181 41.020 −6.168 20.712 1.00 0.02 ATOM 1601 CB THR A 181 39.553 −6.503 20.935 1.00 0.65 ATOM 1602 OG1 THR A 181 38.817 −5.290 20.839 1.00 1.46 ATOM 1603 CG2 THR A 181 39.298 −7.129 22.301 1.00 0.43 ATOM 1604 C THR A 181 41.786 −7.392 20.221 1.00 0.03 ATOM 1605 O THR A 181 41.690 −7.780 19.049 1.00 0.01 ATOM 1606 N ASP A 182 42.579 −7.975 21.104 1.00 0.02 ATOM 1608 CA ASP A 182 43.355 −9.159 20.722 1.00 0.02 ATOM 1609 CB ASP A 182 44.572 −9.307 21.629 1.00 0.71 ATOM 1610 CG ASP A 182 45.377 −10.519 21.174 1.00 0.75 ATOM 1611 OD1 ASP A 182 45.596 −10.586 19.972 1.00 0.69 ATOM 1612 OD2 ASP A 182 45.339 −11.498 21.906 1.00 0.90 ATOM 1613 C ASP A 182 42.503 −10.426 20.768 1.00 0.00 ATOM 1614 O ASP A 182 42.490 −11.169 21.756 1.00 0.02 ATOM 1615 N PHE A 183 42.021 −10.785 19.589 1.00 0.02 ATOM 1617 CA PHE A 183 41.163 −11.961 19.427 1.00 0.01 ATOM 1618 CB PHE A 183 40.225 −11.721 18.253 1.00 0.02 ATOM 1619 CG PHE A 183 39.191 −10.636 18.536 1.00 0.01 ATOM 1620 CD1 PHE A 183 38.500 −10.639 19.740 1.00 0.01 ATOM 1621 CE1 PHE A 183 37.560 −9.652 20.004 1.00 0.01 ATOM 1622 CZ PHE A 183 37.310 −8.664 19.061 1.00 0.02 ATOM 1623 CE2 PHE A 183 37.996 −8.666 17.855 1.00 0.00 ATOM 1624 CD2 PHE A 183 38.936 −9.652 17.593 1.00 0.02 ATOM 1625 C PHE A 183 41.938 −13.264 19.246 1.00 0.01 ATOM 1626 O PHE A 183 41.325 −14.335 19.184 1.00 0.01 ATOM 1627 N SER A 184 43.263 −13.192 19.291 1.00 0.01 ATOM 1629 CA SER A 184 44.070 −14.418 19.321 1.00 0.02 ATOM 1630 CB SER A 184 45.492 −14.127 18.858 1.00 0.40 ATOM 1631 OG SER A 184 46.142 −13.367 19.866 1.00 1.07 ATOM 1632 C SER A 184 44.093 −14.982 20.744 1.00 0.02 ATOM 1633 O SER A 184 44.506 −16.127 20.961 1.00 0.01 ATOM 1634 N ASN A 185 43.690 −14.163 21.703 1.00 0.00 ATOM 1636 CA ASN A 185 43.336 −14.670 23.023 1.00 0.02 ATOM 1637 CB ASN A 185 44.304 −14.136 24.072 1.00 0.49 ATOM 1638 CG ASN A 185 44.036 −14.815 25.415 1.00 1.32 ATOM 1639 OD1 ASN A 185 42.918 −14.767 25.947 1.00 1.55 ATOM 1640 ND2 ASN A 185 45.056 −15.472 25.933 1.00 2.05 ATOM 1643 C ASN A 185 41.915 −14.218 23.337 1.00 0.02 ATOM 1644 O ASN A 185 41.708 −13.214 24.036 1.00 0.02 ATOM 1645 N PRO A 186 40.958 −15.073 23.010 1.00 0.01 ATOM 1646 CA PRO A 186 39.547 −14.699 23.123 1.00 0.02 ATOM 1647 CB PRO A 186 38.808 −15.742 22.343 1.00 0.14 ATOM 1648 CG PRO A 186 39.772 −16.852 21.951 1.00 0.20 ATOM 1649 CD PRO A 186 41.142 −16.410 22.435 1.00 0.14 ATOM 1650 C PRO A 186 39.027 −14.640 24.564 1.00 0.02 ATOM 1651 O PRO A 186 38.000 −13.994 24.797 1.00 0.01 ATOM 1652 N SER A 187 39.786 −15.119 25.538 1.00 0.01 ATOM 1654 CA SER A 187 39.295 −15.048 26.913 1.00 0.02 ATOM 1655 CB SER A 187 39.850 −16.206 27.741 1.00 0.10 ATOM 1656 OG SER A 187 41.267 −16.119 27.793 1.00 1.03 ATOM 1657 C SER A 187 39.656 −13.702 27.533 1.00 0.02 ATOM 1658 O SER A 187 38.778 −13.067 28.128 1.00 0.01 ATOM 1659 N ILE A 188 40.787 −13.144 27.130 1.00 0.02 ATOM 1661 CA ILE A 188 41.173 −11.823 27.627 1.00 0.02 ATOM 1662 CB ILE A 188 42.691 −11.705 27.593 1.00 0.16 ATOM 1663 CG2 ILE A 188 43.138 −10.302 27.988 1.00 0.71 ATOM 1664 CG1 ILE A 188 43.322 −12.737 28.518 1.00 0.98 ATOM 1665 CD1 ILE A 188 44.841 −12.619 28.524 1.00 1.50 ATOM 1666 C ILE A 188 40.537 −10.730 26.782 1.00 0.02 ATOM 1667 O ILE A 188 40.068 −9.726 27.338 1.00 0.02 ATOM 1668 N ALA A 189 40.245 −11.065 25.536 1.00 0.02 ATOM 1670 CA ALA A 189 39.522 −10.135 24.668 1.00 0.02 ATOM 1671 CB ALA A 189 39.525 −10.700 23.255 1.00 0.02 ATOM 1672 C ALA A 189 38.085 −9.964 25.141 1.00 0.02 ATOM 1673 O ALA A 189 37.648 −8.831 25.380 1.00 0.02 ATOM 1674 N GLN A 190 37.508 −11.065 25.592 1.00 0.01 ATOM 1676 CA GLN A 190 36.132 −11.066 26.081 1.00 0.02 ATOM 1677 CB GLN A 190 35.692 −12.514 26.029 1.00 0.17 ATOM 1678 CG GLN A 190 34.203 −12.707 26.211 1.00 0.67 ATOM 1679 CD GLN A 190 33.898 −14.128 25.770 1.00 1.38 ATOM 1680 OE1 GLN A 190 32.823 −14.394 25.226 1.00 2.17 ATOM 1681 NE2 GLN A 190 34.948 −14.929 25.720 1.00 2.05 ATOM 1684 C GLN A 190 36.026 −10.537 27.506 1.00 0.02 ATOM 1685 O GLN A 190 35.015 −9.914 27.853 1.00 0.01 ATOM 1686 N ALA A 191 37.130 −10.569 28.234 1.00 0.01 ATOM 1688 CA ALA A 191 37.151 −9.978 29.568 1.00 0.02 ATOM 1689 CB ALA A 191 38.387 −10.467 30.312 1.00 0.09 ATOM 1690 C ALA A 191 37.178 −8.460 29.464 1.00 0.02 ATOM 1691 O ALA A 191 36.374 −7.803 30.135 1.00 0.01 ATOM 1692 N ARG A 192 37.836 −7.949 28.435 1.00 0.01 ATOM 1694 CA ARG A 192 37.847 −6.502 28.210 1.00 0.01 ATOM 1695 CB ARG A 192 38.983 −6.166 27.256 1.00 0.18 ATOM 1696 CG ARG A 192 40.332 −6.317 27.948 1.00 0.97 ATOM 1697 CD ARG A 192 41.484 −6.059 26.985 1.00 1.10 ATOM 1698 NE ARG A 192 41.578 −7.133 25.987 1.00 1.92 ATOM 1699 CZ ARG A 192 42.740 −7.521 25.458 1.00 2.61 ATOM 1700 NH1 ARG A 192 43.863 −6.868 25.763 1.00 3.27 ATOM 1701 NH2 ARG A 192 42.773 −8.534 24.592 1.00 3.22 ATOM 1702 C ARG A 192 36.524 −6.006 27.636 1.00 0.02 ATOM 1703 O ARG A 192 36.009 −4.985 28.111 1.00 0.01 ATOM 1704 N ILE A 193 35.862 −6.846 26.857 1.00 0.01 ATOM 1706 CA ILE A 193 34.552 −6.478 26.313 1.00 0.02 ATOM 1707 CB ILE A 193 34.160 −7.505 25.258 1.00 0.01 ATOM 1708 CG2 ILE A 193 32.765 −7.216 24.719 1.00 0.02 ATOM 1709 CG1 ILE A 193 35.172 −7.510 24.121 1.00 0.02 ATOM 1710 CD1 ILE A 193 34.843 −8.578 23.087 1.00 0.02 ATOM 1711 C ILE A 193 33.486 −6.430 27.406 1.00 0.00 ATOM 1712 O ILE A 193 32.863 −5.376 27.594 1.00 0.02 ATOM 1713 N ASN A 194 33.515 −7.409 28.296 1.00 0.02 ATOM 1715 CA ASN A 194 32.533 −7.467 29.378 1.00 0.02 ATOM 1716 CB ASN A 194 32.502 −8.893 29.900 1.00 0.02 ATOM 1717 CG ASN A 194 31.780 −9.764 28.883 1.00 0.02 ATOM 1718 OD1 ASN A 194 30.787 −9.320 28.289 1.00 0.02 ATOM 1719 ND2 ASN A 194 32.154 −11.028 28.844 1.00 0.02 ATOM 1722 C ASN A 194 32.821 −6.502 30.521 1.00 0.02 ATOM 1723 O ASN A 194 31.865 −6.020 31.147 1.00 0.02 ATOM 1724 N SER A 195 34.059 −6.057 30.644 1.00 0.02 ATOM 1726 CA SER A 195 34.377 −5.051 31.654 1.00 0.02 ATOM 1727 CB SER A 195 35.849 −5.156 32.027 1.00 0.14 ATOM 1728 OG SER A 195 36.057 −6.438 32.601 1.00 0.40 ATOM 1729 C SER A 195 34.074 −3.648 31.148 1.00 0.01 ATOM 1730 O SER A 195 33.677 −2.794 31.947 1.00 0.02 ATOM 1731 N HIS A 196 34.015 −3.488 29.836 1.00 0.02 ATOM 1733 CA HIS A 196 33.628 −2.201 29.258 1.00 0.02 ATOM 1734 CB HIS A 196 34.143 −2.145 27.826 1.00 0.17 ATOM 1735 CG HIS A 196 33.764 −0.873 27.099 1.00 1.00 ATOM 1736 ND1 HIS A 196 32.882 −0.755 26.088 1.00 2.01 ATOM 1738 CE1 HIS A 196 32.814 0.536 25.706 1.00 2.59 ATOM 1739 NE2 HIS A 196 33.661 1.240 26.490 1.00 2.37 ATOM 1740 GD2 HIS A 196 34.254 0.385 27.353 1.00 1.67 ATOM 1741 C HIS A 196 32.111 −2.043 29.265 1.00 0.03 ATOM 1742 O HIS A 196 31.601 −0.956 29.572 1.00 0.02 ATOM 1743 N VAL A 197 31.420 −3.169 29.187 1.00 0.01 ATOM 1745 CA VAL A 197 29.958 −3.169 29.286 1.00 0.02 ATOM 1746 CB VAL A 197 29.467 −4.552 28.875 1.00 0.01 ATOM 1747 CG1 VAL A 197 27.989 −4.721 29.171 1.00 0.01 ATOM 1748 CG2 VAL A 197 29.752 −4.831 27.404 1.00 0.01 ATOM 1749 C VAL A 197 29.518 −2.860 30.716 1.00 0.01 ATOM 1750 O VAL A 197 28.743 −1.915 30.935 1.00 0.01 ATOM 1751 N LYS A 198 30.263 −3.415 31.658 1.00 0.01 ATOM 1753 CA LYS A 198 30.030 −3.139 33.072 1.00 0.01 ATOM 1754 CB LYS A 198 30.907 −4.106 33.854 1.00 0.11 ATOM 1755 CG LYS A 198 30.931 −3.796 35.343 1.00 0.81 ATOM 1756 CD LYS A 198 31.958 −4.676 36.041 1.00 0.83 ATOM 1757 CE LYS A 198 33.334 −4.491 35.411 1.00 1.41 ATOM 1758 NZ LYS A 198 34.329 −5.373 36.041 1.00 2.32 ATOM 1759 C LYS A 198 30.403 −1.705 33.439 1.00 0.01 ATOM 1760 O LYS A 198 29.614 −1.038 34.119 1.00 0.01 ATOM 1761 N LYS A 199 31.413 −1.162 32.779 1.00 0.01 ATOM 1763 CA LYS A 199 31.881 0.196 33.062 1.00 0.02 ATOM 1764 CB LYS A 199 33.240 0.367 32.391 1.00 0.02 ATOM 1765 CG LYS A 199 33.799 1.771 32.582 1.00 0.02 ATOM 1766 CD LYS A 199 35.105 1.951 31.818 1.00 0.01 ATOM 1767 CE LYS A 199 35.636 3.373 31.955 1.00 0.02 ATOM 1768 NZ LYS A 199 36.877 3.548 31.184 1.00 0.01 ATOM 1769 C LYS A 199 30.935 1.281 32.553 1.00 0.02 ATOM 1770 O LYS A 199 30.631 2.207 33.317 1.00 0.02 ATOM 1771 N LYS A 200 30.300 1.076 31.409 1.00 0.01 ATOM 1773 CA LYS A 200 29.396 2.122 30.913 1.00 0.00 ATOM 1774 CB LYS A 200 29.241 2.021 29.403 1.00 0.54 ATOM 1775 CG LYS A 200 30.566 2.268 28.696 1.00 0.77 ATOM 1776 CD LYS A 200 30.344 2.585 27.223 1.00 1.41 ATOM 1777 CE LYS A 200 29.590 3.900 27.054 1.00 2.57 ATOM 1778 NZ LYS A 200 29.363 4.207 25.632 1.00 3.36 ATOM 1779 C LYS A 200 28.025 2.062 31.577 1.00 0.02 ATOM 1780 O LYS A 200 27.350 3.091 31.696 1.00 0.01 ATOM 1781 N THR A 201 27.706 0.918 32.158 1.00 0.01 ATOM 1783 CA THR A 201 26.471 0.794 32.935 1.00 0.02 ATOM 1784 CB THR A 201 25.869 −0.573 32.675 1.00 0.02 ATOM 1785 OG1 THR A 201 26.737 −1.537 33.256 1.00 0.01 ATOM 1786 CG2 THR A 201 25.729 −0.843 31.182 1.00 0.02 ATOM 1787 C THR A 201 26.710 0.937 34.436 1.00 0.01 ATOM 1788 O THR A 201 25.821 0.578 35.217 1.00 0.02 ATOM 1789 N GLN A 202 27.929 1.298 34.815 1.00 0.01 ATOM 1791 CA GLN A 202 28.342 1.444 36.222 1.00 0.01 ATOM 1792 CB GLN A 202 27.749 2.737 36.767 1.00 0.01 ATOM 1793 CG GLN A 202 28.390 3.955 36.107 1.00 0.02 ATOM 1794 CD GLN A 202 29.832 4.128 36.586 1.00 0.02 ATOM 1795 OE1 GLN A 202 30.071 4.474 37.748 1.00 0.02 ATOM 1796 NE2 GLN A 202 30.781 3.868 35.702 1.00 0.02 ATOM 1799 C GLN A 202 27.944 0.258 37.096 1.00 0.02 ATOM 1800 O GLN A 202 27.133 0.391 38.017 1.00 0.02 ATOM 1801 N GLY A 203 28.388 −0.919 36.684 1.00 0.01 ATOM 1803 CA GLY A 203 28.146 −2.150 37.443 1.00 0.02 ATOM 1804 C GLY A 203 26.858 −2.892 37.073 1.00 0.01 ATOM 1805 O GLY A 203 26.715 −4.067 37.432 1.00 0.02 ATOM 1806 N LYS A 204 25.948 −2.241 36.365 1.00 0.02 ATOM 1808 CA LYS A 204 24.640 −2.848 36.097 1.00 0.02 ATOM 1809 CB LYS A 204 23.717 −1.784 35.509 1.00 0.02 ATOM 1810 CG LYS A 204 22.306 −2.319 35.295 1.00 0.02 ATOM 1811 CD LYS A 204 21.668 −2.736 36.615 1.00 0.01 ATOM 1812 CE LYS A 204 20.310 −3.385 36.387 1.00 0.02 ATOM 1813 NZ LYS A 204 20.448 −4.592 35.555 1.00 0.01 ATOM 1814 C LYS A 204 24.714 −4.046 35.154 1.00 0.02 ATOM 1815 O LYS A 204 24.300 −5.148 35.535 1.00 0.01 ATOM 1816 N VAL A 205 25.316 −3.888 33.988 1.00 0.02 ATOM 1818 CA VAL A 205 25.328 −5.002 33.046 1.00 0.02 ATOM 1819 CB VAL A 205 24.963 −4.480 31.663 1.00 1.75 ATOM 1820 CG1 VAL A 205 24.930 −5.618 30.660 1.00 2.29 ATOM 1821 CG2 VAL A 205 23.609 −3.781 31.690 1.00 1.90 ATOM 1822 C VAL A 205 26.693 −5.678 33.049 1.00 0.02 ATOM 1823 O VAL A 205 27.606 −5.332 32.287 1.00 0.02 ATOM 1824 N VAL A 206 26.830 −6.623 33.959 1.00 0.02 ATOM 1826 CA VAL A 206 28.087 −7.359 34.091 1.00 0.02 ATOM 1827 CB VAL A 206 28.282 −7.714 35.564 1.00 0.02 ATOM 1828 CG1 VAL A 206 28.669 −6.487 36.378 1.00 0.02 ATOM 1829 CG2 VAL A 206 27.044 −8.374 36.164 1.00 0.02 ATOM 1830 C VAL A 206 28.135 −8.619 33.226 1.00 0.02 ATOM 1831 O VAL A 206 27.325 −9.541 33.380 1.00 0.02 ATOM 1832 N ASP A 207 29.094 −8.621 32.311 1.00 0.01 ATOM 1834 CA ASP A 207 29.461 −9.822 31.538 1.00 0.01 ATOM 1835 CB ASP A 207 29.973 −10.855 32.545 1.00 0.01 ATOM 1836 CG ASP A 207 30.574 −12.075 31.861 1.00 0.02 ATOM 1837 OD1 ASP A 207 31.781 −12.066 31.656 1.00 0.02 ATOM 1838 OD2 ASP A 207 29.828 −13.006 31.589 1.00 0.01 ATOM 1839 C ASP A 207 28.307 −10.405 30.715 1.00 0.02 ATOM 1840 O ASP A 207 27.567 −11.272 31.197 1.00 0.01 ATOM 1841 N ILE A 208 28.194 −9.988 29.464 1.00 0.02 ATOM 1843 CA ILE A 208 27.103 −10.495 28.619 1.00 0.02 ATOM 1844 CB ILE A 208 26.250 −9.320 28.151 1.00 2.45 ATOM 1845 CG2 ILE A 208 25.292 −9.714 27.031 1.00 3.34 ATOM 1846 CG1 ILE A 208 25.463 −8.770 29.330 1.00 3.29 ATOM 1847 CD1 ILE A 208 24.554 −9.843 29.922 1.00 4.19 ATOM 1848 C ILE A 208 27.613 −11.320 27.438 1.00 0.01 ATOM 1849 O ILE A 208 26.952 −12.280 27.023 1.00 0.00 ATOM 1850 N ILE A 209 28.838 −11.054 27.020 1.00 0.02 ATOM 1852 CA ILE A 209 29.452 −11.827 25.933 1.00 0.02 ATOM 1853 CB ILE A 209 30.602 −10.999 25.362 1.00 1.56 ATOM 1854 CG2 ILE A 209 31.305 −11.731 24.229 1.00 2.38 ATOM 1855 CG1 ILE A 209 30.097 −9.651 24.861 1.00 2.02 ATOM 1856 CD1 ILE A 209 29.197 −9.802 23.639 1.00 2.14 ATOM 1857 C ILE A 209 29.949 −13.157 26.494 1.00 0.02 ATOM 1858 O ILE A 209 30.876 −13.182 27.314 1.00 0.02 ATOM 1859 N GLN A 210 29.245 −14.223 26.140 1.00 0.02 ATOM 1861 CA GLN A 210 29.515 −15.557 26.698 1.00 0.01 ATOM 1862 CB GLN A 210 28.181 −16.245 26.979 1.00 1.43 ATOM 1863 CG GLN A 210 27.267 −15.429 27.887 1.00 2.09 ATOM 1864 CD GLN A 210 27.915 −15.196 29.247 1.00 2.72 ATOM 1865 OE1 GLN A 210 28.581 −16.080 29.799 1.00 2.94 ATOM 1866 NE2 GLN A 210 27.649 −14.026 29.797 1.00 3.33 ATOM 1869 C GLN A 210 30.311 −16.465 25.763 1.00 0.02 ATOM 1870 O GLN A 210 30.722 −17.560 26.167 1.00 0.02 ATOM 1871 N GLY A 211 30.544 −16.021 24.542 1.00 0.01 ATOM 1873 CA GLY A 211 31.234 −16.887 23.587 1.00 0.02 ATOM 1874 C GLY A 211 31.664 −16.167 22.315 1.00 0.01 ATOM 1875 O GLY A 211 30.924 −16.144 21.323 1.00 0.01 ATOM 1876 N LEU A 212 32.829 −15.544 22.365 1.00 0.02 ATOM 1878 CA LEU A 212 33.429 −15.021 21.131 1.00 0.02 ATOM 1879 CB LEU A 212 34.730 −14.291 21.442 1.00 0.02 ATOM 1880 CG LEU A 212 34.509 −12.973 22.166 1.00 0.03 ATOM 1881 CD1 LEU A 212 35.848 −12.284 22.399 1.00 0.02 ATOM 1882 CD2 LEU A 212 33.589 −12.070 21.357 1.00 0.02 ATOM 1883 C LEU A 212 33.764 −16.166 20.184 1.00 0.02 ATOM 1884 O LEU A 212 34.391 −17.152 20.586 1.00 0.02 ATOM 1885 N ASP A 213 33.348 −16.028 18.937 1.00 0.02 ATOM 1887 CA ASP A 213 33.725 −17.009 17.915 1.00 0.02 ATOM 1888 CB ASP A 213 33.025 −16.663 16.602 1.00 0.02 ATOM 1889 CG ASP A 213 31.513 −16.871 16.720 1.00 0.02 ATOM 1890 OD1 ASP A 213 31.146 −17.786 17.446 1.00 0.02 ATOM 1891 OD2 ASP A 213 30.814 −16.339 15.873 1.00 0.01 ATOM 1892 C ASP A 213 35.243 −17.007 17.735 1.00 0.01 ATOM 1893 O ASP A 213 35.895 −15.971 17.900 1.00 0.02 ATOM 1894 N LEU A 214 35.801 −18.139 17.333 1.00 0.02 ATOM 1896 CA LEU A 214 37.269 −18.245 17.212 1.00 0.02 ATOM 1897 CB LEU A 214 37.656 −19.719 17.218 1.00 0.71 ATOM 1898 CG LEU A 214 37.303 −20.384 18.545 1.00 1.18 ATOM 1899 CD1 LEU A 214 37.561 −21.886 18.490 1.00 2.00 ATOM 1900 CD2 LEU A 214 38.070 −19.749 19.702 1.00 0.70 ATOM 1901 C LEU A 214 37.834 −17.570 15.958 1.00 0.02 ATOM 1902 O LEU A 214 39.046 −17.360 15.853 1.00 0.01 ATOM 1903 N LEU A 215 36.952 −17.175 15.054 1.00 0.02 ATOM 1905 CA LEU A 215 37.337 −16.379 13.885 1.00 0.01 ATOM 1906 CB LEU A 215 36.730 −17.010 12.636 1.00 0.36 ATOM 1907 CG LEU A 215 37.280 −18.410 12.386 1.00 0.58 ATOM 1908 CD1 LEU A 215 36.543 −19.089 11.238 1.00 0.90 ATOM 1909 CD2 LEU A 215 38.782 −18.375 12.118 1.00 0.75 ATOM 1910 C LEU A 215 36.851 −14.934 14.019 1.00 0.02 ATOM 1911 O LEU A 215 36.600 −14.274 13.002 1.00 0.02 ATOM 1912 N THR A 216 36.624 −14.491 15.248 1.00 0.02 ATOM 1914 CA THR A 216 36.066 −13.154 15.478 1.00 0.01 ATOM 1915 CB THR A 216 35.668 −13.016 16.948 1.00 0.02 ATOM 1916 OG1 THR A 216 34.418 −13.675 17.093 1.00 0.02 ATOM 1917 CG2 THR A 216 35.435 −11.568 17.361 1.00 0.02 ATOM 1918 C THR A 216 36.995 −12.024 15.054 1.00 0.01 ATOM 1919 O THR A 216 38.106 −11.858 15.566 1.00 0.02 ATOM 1920 N ALA A 217 36.546 −11.310 14.035 1.00 0.02 ATOM 1922 CA ALA A 217 37.233 −10.105 13.595 1.00 0.01 ATOM 1923 CB ALA A 217 37.410 −10.187 12.085 1.00 0.41 ATOM 1924 C ALA A 217 36.467 −8.837 13.977 1.00 0.02 ATOM 1925 O ALA A 217 37.029 −7.735 13.887 1.00 0.02 ATOM 1926 N MET A 218 35.230 −8.988 14.433 1.00 0.02 ATOM 1928 CA MET A 218 34.427 −7.813 14.799 1.00 0.02 ATOM 1929 CB MET A 218 33.769 −7.272 13.532 1.00 0.89 ATOM 1930 CG MET A 218 32.877 −6.070 13.823 1.00 1.03 ATOM 1931 SD MET A 218 33.726 −4.594 14.425 1.00 1.41 ATOM 1932 CE MET A 218 34.664 −4.210 12.930 1.00 1.60 ATOM 1933 C MET A 218 33.340 −8.122 15.832 1.00 0.01 ATOM 1934 O MET A 218 32.415 −8.910 15.578 1.00 0.01 ATOM 1935 N VAL A 219 33.482 −7.517 16.999 1.00 0.02 ATOM 1937 CA VAL A 219 32.422 −7.542 18.017 1.00 0.02 ATOM 1938 CB VAL A 219 33.068 −7.764 19.381 1.00 0.06 ATOM 1939 CG1 VAL A 219 32.055 −7.782 20.521 1.00 0.12 ATOM 1940 CG2 VAL A 219 33.866 −9.056 19.379 1.00 0.07 ATOM 1941 C VAL A 219 31.650 −6.220 17.995 1.00 0.01 ATOM 1942 O VAL A 219 32.250 −5.139 17.968 1.00 0.02 ATOM 1943 N LEU A 220 30.333 −6.315 18.009 1.00 0.01 ATOM 1945 CA LEU A 220 29.482 −5.123 17.957 1.00 0.01 ATOM 1946 CB LEU A 220 28.642 −5.205 16.680 1.00 0.01 ATOM 1947 CG LEU A 220 28.089 −3.862 16.191 1.00 0.01 ATOM 1948 CD1 LEU A 220 27.668 −3.953 14.729 1.00 0.01 ATOM 1949 CD2 LEU A 220 26.946 −3.313 17.041 1.00 0.01 ATOM 1950 C LEU A 220 28.594 −5.077 19.200 1.00 0.01 ATOM 1951 O LEU A 220 27.497 −5.650 19.218 1.00 0.01 ATOM 1952 N VAL A 221 29.073 −4.388 20.221 1.00 0.01 ATOM 1954 CA VAL A 221 28.305 −4.223 21.462 1.00 0.01 ATOM 1955 CB VAL A 221 29.305 −4.000 22.595 1.00 0.02 ATOM 1956 CG1 VAL A 221 28.634 −3.684 23.928 1.00 0.01 ATOM 1957 CG2 VAL A 221 30.216 −5.211 22.736 1.00 0.02 ATOM 1958 C VAL A 221 27.345 −3.041 21.336 1.00 0.01 ATOM 1959 O VAL A 221 27.729 −1.976 20.839 1.00 0.02 ATOM 1960 N ASN A 222 26.090 −3.262 21.689 1.00 0.01 ATOM 1962 CA ASN A 222 25.084 −2.195 21.631 1.00 0.01 ATOM 1963 CB ASN A 222 24.364 −2.314 20.295 1.00 0.01 ATOM 1964 CG ASN A 222 23.343 −1.197 20.131 1.00 0.01 ATOM 1965 OD1 ASN A 222 22.132 −1.435 20.262 1.00 0.01 ATOM 1966 ND2 ASN A 222 23.834 0.015 19.928 1.00 0.01 ATOM 1969 C ASN A 222 24.095 −2.302 22.795 1.00 0.01 ATOM 1970 O ASN A 222 22.975 −2.808 22.661 1.00 0.02 ATOM 1971 N HIS A 223 24.526 −1.833 23.950 1.00 0.01 ATOM 1973 CA HIS A 223 23.697 −1.944 25.158 1.00 0.02 ATOM 1974 CB HIS A 223 24.551 −2.496 26.298 1.00 0.87 ATOM 1975 CG HIS A 223 24.966 −3.960 26.176 1.00 1.28 ATOM 1976 ND1 HIS A 223 25.150 −4.803 27.206 1.00 1.65 ATOM 1978 CE1 HIS A 223 25.513 −6.015 26.741 1.00 2.67 ATOM 1979 NE2 HIS A 223 25.558 −5.941 25.393 1.00 3.07 ATOM 1980 CD2 HIS A 223 25.223 −4.684 25.032 1.00 2.44 ATOM 1981 C HIS A 223 23.079 −0.594 25.527 1.00 0.01 ATOM 1982 O HIS A 223 23.507 0.440 24.998 1.00 0.01 ATOM 1983 N ILE A 224 22.034 −0.619 26.344 1.00 0.01 ATOM 1985 CA ILE A 224 21.324 0.625 26.704 1.00 0.02 ATOM 1986 CB ILE A 224 20.522 1.038 25.466 1.00 0.01 ATOM 1987 CG2 ILE A 224 19.655 −0.104 24.944 1.00 0.01 ATOM 1988 CG1 ILE A 224 19.686 2.287 25.693 1.00 0.01 ATOM 1989 CD1 ILE A 224 18.973 2.688 24.405 1.00 0.00 ATOM 1990 C ILE A 224 20.434 0.501 27.963 1.00 0.01 ATOM 1991 O ILE A 224 19.450 −0.251 27.990 1.00 0.02 ATOM 1992 N PHE A 225 20.793 1.254 28.996 1.00 0.02 ATOM 1994 CA PHE A 225 20.052 1.255 30.279 1.00 0.01 ATOM 1995 CB PHE A 225 21.077 1.048 31.398 1.00 0.01 ATOM 1996 CG PHE A 225 20.524 1.137 32.821 1.00 0.02 ATOM 1997 CD1 PHE A 225 19.720 0.121 33.320 1.00 0.00 ATOM 1998 CE1 PHE A 225 19.207 0.206 34.608 1.00 0.01 ATOM 1999 CZ PHE A 225 19.500 1.309 35.400 1.00 0.01 ATOM 2000 CE2 PHE A 225 20.306 2.324 34.903 1.00 0.02 ATOM 2001 CD2 PHE A 225 20.817 2.238 33.615 1.00 0.01 ATOM 2002 C PHE A 225 19.257 2.550 30.534 1.00 0.02 ATOM 2003 O PHE A 225 19.752 3.660 30.299 1.00 0.02 ATOM 2004 N PHE A 226 18.034 2.401 31.023 1.00 0.01 ATOM 2006 CA PHE A 226 17.212 3.560 31.412 1.00 0.01 ATOM 2007 CB PHE A 226 16.186 3.802 30.308 1.00 0.01 ATOM 2008 CG PHE A 226 15.325 5.054 30.479 1.00 0.01 ATOM 2009 CD1 PHE A 226 15.909 6.259 30.851 1.00 0.01 ATOM 2010 CE1 PHE A 226 15.124 7.394 31.007 1.00 0.01 ATOM 2011 CZ PHE A 226 13.756 7.327 30.783 1.00 0.01 ATOM 2012 CE2 PHE A 226 13.174 6.126 30.399 1.00 0.01 ATOM 2013 CD2 PHE A 226 13.959 4.992 30.243 1.00 0.01 ATOM 2014 C PHE A 226 16.482 3.348 32.748 1.00 0.02 ATOM 2015 O PHE A 226 15.952 2.263 33.022 1.00 0.02 ATOM 2016 N LYS A 227 16.510 4.367 33.594 1.00 0.01 ATOM 2018 CA LYS A 227 15.692 4.371 34.819 1.00 0.01 ATOM 2019 CB LYS A 227 16.587 4.677 36.016 1.00 0.01 ATOM 2020 CG LYS A 227 15.844 4.470 37.332 1.00 0.02 ATOM 2021 CD LYS A 227 16.766 4.616 38.535 1.00 0.00 ATOM 2022 CE LYS A 227 16.032 4.290 39.831 1.00 0.02 ATOM 2023 NZ LYS A 227 16.933 4.396 40.990 1.00 0.01 ATOM 2024 C LYS A 227 14.570 5.412 34.690 1.00 0.01 ATOM 2025 O LYS A 227 14.826 6.574 34.352 1.00 0.00 ATOM 2026 N ALA A 228 13.343 4.984 34.942 1.00 0.02 ATOM 2028 CA ALA A 228 12.164 5.833 34.703 1.00 0.00 ATOM 2029 CB ALA A 228 10.949 4.915 34.631 1.00 2.74 ATOM 2030 C ALA A 228 11.903 6.930 35.745 1.00 0.02 ATOM 2031 O ALA A 228 11.520 6.656 36.890 1.00 0.01 ATOM 2032 N LYS A 229 12.091 8.173 35.323 1.00 0.02 ATOM 2034 CA LYS A 229 11.680 9.329 36.140 1.00 0.01 ATOM 2035 CB LYS A 229 12.712 10.446 36.047 1.00 0.01 ATOM 2036 CG LYS A 229 14.112 10.023 36.464 1.00 0.02 ATOM 2037 CD LYS A 229 15.020 11.247 36.501 1.00 0.01 ATOM 2038 CE LYS A 229 14.956 12.014 35.184 1.00 0.02 ATOM 2039 NZ LYS A 229 15.754 13.250 35.241 1.00 0.02 ATOM 2040 C LYS A 229 10.359 9.894 35.625 1.00 0.02 ATOM 2041 O LYS A 229 10.340 10.602 34.614 1.00 0.01 ATOM 2042 N TRP A 230 9.288 9.641 36.355 1.00 0.01 ATOM 2044 CA TRP A 230 7.947 10.079 35.942 1.00 0.00 ATOM 2045 CB TRP A 230 6.928 9.244 36.711 1.00 0.01 ATOM 2046 CG TRP A 230 7.132 7.747 36.623 1.00 0.02 ATOM 2047 CD1 TRP A 230 7.766 6.944 37.544 1.00 0.02 ATOM 2048 NE1 TRP A 230 7.738 5.666 37.093 1.00 0.02 ATOM 2050 CE2 TRP A 230 7.105 5.583 35.910 1.00 0.02 ATOM 2051 CZ2 TRP A 230 6.833 4.520 35.061 1.00 0.02 ATOM 2052 CH2 TRP A 230 6.138 4.745 33.880 1.00 0.02 ATOM 2053 CZ3 TRP A 230 5.714 6.027 33.543 1.00 0.02 ATOM 2054 CE3 TRP A 230 5.985 7.098 34.386 1.00 0.01 ATOM 2055 CD2 TRP A 230 6.681 6.878 35.563 1.00 0.01 ATOM 2056 C TRP A 230 7.682 11.542 36.280 1.00 0.01 ATOM 2057 O TRP A 230 8.238 12.071 37.246 1.00 0.02 ATOM 2058 N GLU A 231 6.838 12.187 35.486 1.00 0.00 ATOM 2060 CA GLU A 231 6.317 13.505 35.880 1.00 0.02 ATOM 2061 CB GLU A 231 5.940 14.317 34.648 1.00 0.01 ATOM 2062 CG GLU A 231 7.167 14.820 33.909 1.00 0.02 ATOM 2063 CD GLU A 231 6.748 15.520 32.625 1.00 0.01 ATOM 2064 OE1 GLU A 231 5.717 15.135 32.085 1.00 0.02 ATOM 2065 OE2 GLU A 231 7.483 16.398 32.189 1.00 0.01 ATOM 2066 C GLU A 231 5.075 13.325 36.743 1.00 0.01 ATOM 2067 O GLU A 231 4.708 14.204 37.531 1.00 0.02 ATOM 2068 N LYS A 232 4.469 12.157 36.611 1.00 0.01 ATOM 2070 CA LYS A 232 3.346 11.766 37.468 1.00 0.00 ATOM 2071 CB LYS A 232 2.213 11.333 36.545 1.00 0.65 ATOM 2072 CG LYS A 232 1.961 12.369 35.451 1.00 1.25 ATOM 2073 CD LYS A 232 0.943 11.872 34.432 1.00 1.89 ATOM 2074 CE LYS A 232 0.755 12.859 33.286 1.00 2.53 ATOM 2075 NZ LYS A 232 −0.202 12.335 32.300 1.00 3.06 ATOM 2076 C LYS A 232 3.774 10.593 38.356 1.00 0.02 ATOM 2077 O LYS A 232 3.609 9.431 37.968 1.00 0.02 ATOM 2078 N PRO A 233 4.328 10.898 39.522 1.00 0.01 ATOM 2079 CA PRO A 233 5.023 9.884 40.325 1.00 0.02 ATOM 2080 CB PRO A 233 5.778 10.650 41.368 1.00 0.02 ATOM 2081 CG PRO A 233 5.390 12.117 41.289 1.00 0.02 ATOM 2082 CD PRO A 233 4.451 12.240 40.101 1.00 0.01 ATOM 2083 C PRO A 233 4.059 8.901 40.978 1.00 0.02 ATOM 2084 O PRO A 233 2.893 9.225 41.240 1.00 0.02 ATOM 2085 N PHE A 234 4.542 7.694 41.205 1.00 0.01 ATOM 2087 CA PHE A 234 3.728 6.687 41.895 1.00 0.02 ATOM 2088 CB PHE A 234 3.968 5.325 41.258 1.00 0.02 ATOM 2089 CG PHE A 234 3.429 5.190 39.837 1.00 0.00 ATOM 2090 CD1 PHE A 234 2.096 4.850 39.647 1.00 0.01 ATOM 2091 CE1 PHE A 234 1.587 4.724 38.363 1.00 0.00 ATOM 2092 CZ PHE A 234 2.411 4.934 37.266 1.00 0.01 ATOM 2093 CE2 PHE A 234 3.746 5.268 37.454 1.00 0.01 ATOM 2094 CD2 PHE A 234 4.256 5.393 38.739 1.00 0.02 ATOM 2095 C PHE A 234 4.066 6.647 43.381 1.00 0.00 ATOM 2096 O PHE A 234 5.224 6.848 43.770 1.00 0.01 ATOM 2097 N HIS A 235 3.063 6.405 44.207 1.00 0.02 ATOM 2099 CA HIS A 235 3.314 6.361 45.648 1.00 0.02 ATOM 2100 CB HIS A 235 2.013 6.519 46.415 1.00 1.04 ATOM 2101 CG HIS A 235 1.497 7.939 46.502 1.00 1.51 ATOM 2102 ND1 HIS A 235 1.991 8.923 47.275 1.00 1.88 ATOM 2104 CE1 HIS A 235 1.267 10.045 47.085 1.00 2.58 ATOM 2105 NE2 HIS A 235 0.298 9.759 46.186 1.00 2.75 ATOM 2106 CD2 HIS A 235 0.426 8.463 45.820 1.00 2.28 ATOM 2107 C HIS A 235 3.982 5.068 46.083 1.00 0.00 ATOM 2108 O HIS A 235 3.392 3.983 46.015 1.00 0.02 ATOM 2109 N LEU A 236 5.091 5.250 46.781 1.00 0.02 ATOM 2111 CA LEU A 236 5.863 4.124 47.320 1.00 0.02 ATOM 2112 CB LEU A 236 7.182 4.676 47.855 1.00 0.19 ATOM 2113 CG LEU A 236 8.000 3.629 48.609 1.00 1.21 ATOM 2114 CD1 LEU A 236 8.409 2.469 47.707 1.00 1.76 ATOM 2115 CD2 LEU A 236 9.232 4.263 49.244 1.00 2.24 ATOM 2116 C LEU A 236 5.112 3.425 48.449 1.00 0.03 ATOM 2117 O LEU A 236 4.952 2.202 48.397 1.00 0.00 ATOM 2118 N GLU A 237 4.351 4.205 49.203 1.00 0.02 ATOM 2120 CA GLU A 237 3.569 3.650 50.314 1.00 0.02 ATOM 2121 CB GLU A 237 3.304 4.726 51.378 1.00 0.45 ATOM 2122 CG GLU A 237 2.108 5.650 51.104 1.00 1.07 ATOM 2123 CD GLU A 237 2.452 6.916 50.324 1.00 1.99 ATOM 2124 OE1 GLU A 237 3.332 6.858 49.468 1.00 2.71 ATOM 2125 OE2 GLU A 237 1.787 7.917 50.546 1.00 2.42 ATOM 2126 C GLU A 237 2.244 3.028 49.856 1.00 0.02 ATOM 2127 O GLU A 237 1.503 2.497 50.688 1.00 0.02 ATOM 2128 N TYR A 238 1.938 3.109 48.569 1.00 0.00 ATOM 2130 CA TYR A 238 0.737 2.466 48.039 1.00 0.02 ATOM 2131 CB TYR A 238 −0.174 3.500 47.384 1.00 0.46 ATOM 2132 CG TYR A 238 −0.787 4.495 48.369 1.00 0.77 ATOM 2133 CD1 TYR A 238 −0.917 5.831 48.014 1.00 1.12 ATOM 2134 CE1 TYR A 238 −1.456 6.742 48.912 1.00 1.60 ATOM 2135 CZ TYR A 238 −1.873 6.310 50.164 1.00 1.80 ATOM 2136 OH TYR A 238 −2.344 7.224 51.080 1.00 2.33 ATOM 2137 CE2 TYR A 238 −1.765 4.971 50.517 1.00 1.57 ATOM 2138 CD2 TYR A 238 −1.225 4.062 49.616 1.00 1.05 ATOM 2139 C TYR A 238 1.119 1.366 47.054 1.00 0.00 ATOM 2140 O TYR A 238 0.241 0.775 46.413 1.00 0.02 ATOM 2141 N THR A 239 2.411 1.101 46.939 1.00 0.02 ATOM 2143 CA THR A 239 2.892 0.016 46.077 1.00 0.00 ATOM 2144 CB THR A 239 4.306 0.340 45.605 1.00 0.02 ATOM 2145 OG1 THR A 239 4.256 1.575 44.907 1.00 0.02 ATOM 2146 CG2 THR A 239 4.853 −0.712 44.644 1.00 0.02 ATOM 2147 C THR A 239 2.869 −1.310 46.835 1.00 0.02 ATOM 2148 O THR A 239 3.879 −1.773 47.378 1.00 0.02 ATOM 2149 N ARG A 240 1.701 −1.926 46.830 1.00 0.02 ATOM 2151 CA ARG A 240 1.484 −3.175 47.567 1.00 0.00 ATOM 2152 CB ARG A 240 0.013 −3.242 47.963 1.00 0.53 ATOM 2153 CG ARG A 240 −0.451 −1.955 48.637 1.00 0.68 ATOM 2154 CD ARG A 240 0.259 −1.704 49.964 1.00 1.22 ATOM 2155 NE ARG A 240 −0.136 −0.396 50.506 1.00 1.14 ATOM 2156 CZ ARG A 240 −0.871 −0.252 51.611 1.00 1.24 ATOM 2157 NH1 ARG A 240 −1.252 −1.327 52.305 1.00 1.79 ATOM 2158 NH2 ARG A 240 −1.204 0.969 52.035 1.00 1.15 ATOM 2159 C ARG A 240 1.806 −4.367 46.681 1.00 0.00 ATOM 2160 O ARG A 240 2.199 −4.194 45.524 1.00 0.00 ATOM 2161 N LYS A 241 1.732 −5.561 47.238 1.00 0.02 ATOM 2163 CA LYS A 241 1.793 −6.760 46.396 1.00 0.02 ATOM 2164 CB LYS A 241 2.509 −7.886 47.126 1.00 0.27 ATOM 2165 CG LYS A 241 3.960 −7.513 47.403 1.00 0.53 ATOM 2166 CD LYS A 241 4.703 −8.662 48.071 1.00 0.54 ATOM 2167 CE LYS A 241 4.032 −9.067 49.378 1.00 1.44 ATOM 2168 NZ LYS A 241 4.749 −10.187 50.010 1.00 1.70 ATOM 2169 C LYS A 241 0.367 −7.161 46.039 1.00 0.02 ATOM 2170 O LYS A 241 −0.196 −8.108 46.602 1.00 0.02 ATOM 2171 N ASN A 242 −0.136 −6.506 45.008 1.00 0.02 ATOM 2173 CA ASN A 242 −1.561 −6.535 44.658 1.00 0.02 ATOM 2174 CB ASN A 242 −1.762 −5.487 43.562 1.00 1.70 ATOM 2175 CG ASN A 242 −3.225 −5.103 43.344 1.00 2.00 ATOM 2176 OD1 ASN A 242 −3.967 −4.883 44.308 1.00 2.32 ATOM 2177 ND2 ASN A 242 −3.654 −5.174 42.095 1.00 2.72 ATOM 2180 C ASN A 242 −2.005 −7.912 44.173 1.00 0.01 ATOM 2181 O ASN A 242 −1.179 −8.820 43.981 1.00 0.01 ATOM 2182 N PHE A 243 −3.317 −8.093 44.170 1.00 1.09 ATOM 2184 CA PHE A 243 −3.948 −9.270 43.575 1.00 1.45 ATOM 2185 CB PHE A 243 −5.448 −9.023 43.445 1.00 1.44 ATOM 2186 CG PHE A 243 −6.191 −9.068 44.777 1.00 2.03 ATOM 2187 CD1 PHE A 243 −6.608 −7.895 45.395 1.00 2.31 ATOM 2188 CE1 PHE A 243 −7.282 −7.952 46.608 1.00 3.15 ATOM 2189 CZ PHE A 243 −7.543 −9.181 47.201 1.00 3.65 ATOM 2190 CE2 PHE A 243 −7.132 −10.353 46.581 1.00 3.50 ATOM 2191 CD2 PHE A 243 −6.457 −10.296 45.368 1.00 2.79 ATOM 2192 C PHE A 243 −3.329 −9.557 42.216 1.00 0.50 ATOM 2193 O PHE A 243 −3.162 −8.676 41.360 1.00 1.04 ATOM 2194 N PRO A 244 −2.972 −10.818 42.071 1.00 0.02 ATOM 2195 CA PRO A 244 −1.824 −11.208 41.251 1.00 0.02 ATOM 2196 CB PRO A 244 −1.664 −12.670 41.499 1.00 2.44 ATOM 2197 CG PRO A 244 −2.608 −13.120 42.597 1.00 2.19 ATOM 2198 CD PRO A 244 −3.365 −11.877 43.005 1.00 1.42 ATOM 2199 C PRO A 244 −1.952 −10.906 39.761 1.00 0.02 ATOM 2200 O PRO A 244 −3.037 −10.632 39.229 1.00 0.02 ATOM 2201 N PHE A 245 −0.795 −10.898 39.123 1.00 0.02 ATOM 2203 CA PHE A 245 −0.678 −10.695 37.682 1.00 0.02 ATOM 2204 CB PHE A 245 0.806 −10.487 37.372 1.00 0.02 ATOM 2205 CG PHE A 245 1.133 −9.809 36.043 1.00 0.01 ATOM 2206 CD1 PHE A 245 1.229 −10.552 34.874 1.00 0.02 ATOM 2207 CE1 PHE A 245 1.524 −9.922 33.672 1.00 0.01 ATOM 2208 CZ PHE A 245 1.728 −8.549 33.638 1.00 0.02 ATOM 2209 CE2 PHE A 245 1.637 −7.806 34.807 1.00 0.01 ATOM 2210 CD2 PHE A 245 1.342 −8.436 36.009 1.00 0.01 ATOM 2211 C PHE A 245 −1.194 −11.935 36.960 1.00 0.02 ATOM 2212 O PHE A 245 −0.890 −13.073 37.344 1.00 0.00 ATOM 2213 N LEU A 246 −1.993 −11.701 35.938 1.00 0.01 ATOM 2215 CA LEU A 246 −2.559 −12.782 35.137 1.00 0.02 ATOM 2216 CB LEU A 246 −3.901 −12.339 34.565 1.00 0.69 ATOM 2217 CG LEU A 246 −4.970 −12.241 35.646 1.00 1.13 ATOM 2218 CD1 LEU A 246 −6.254 −11.634 35.093 1.00 1.59 ATOM 2219 CD2 LEU A 246 −5.247 −13.611 36.252 1.00 1.37 ATOM 2220 C LEU A 246 −1.623 −13.147 33.996 1.00 0.02 ATOM 2221 O LEU A 246 −1.581 −12.479 32.953 1.00 0.01 ATOM 2222 N VAL A 247 −0.891 −14.228 34.206 1.00 0.01 ATOM 2224 CA VAL A 247 −0.023 −14.756 33.151 1.00 0.02 ATOM 2225 CB VAL A 247 1.315 −15.191 33.749 1.00 0.02 ATOM 2226 CG1 VAL A 247 1.981 −14.005 34.430 1.00 0.01 ATOM 2227 CG2 VAL A 247 1.180 −16.337 34.742 1.00 0.01 ATOM 2228 C VAL A 247 −0.762 −15.897 32.457 1.00 0.01 ATOM 2229 O VAL A 247 −0.391 −16.365 31.374 1.00 0.00 ATOM 2230 N GLY A 248 −1.841 −16.299 33.106 1.00 0.01 ATOM 2232 CA GLY A 248 −2.850 −17.181 32.524 1.00 0.02 ATOM 2233 C GLY A 248 −4.164 −16.776 33.173 1.00 0.01 ATOM 2234 O GLY A 248 −4.136 −16.141 34.233 1.00 0.02 ATOM 2235 N GLU A 249 −5.279 −17.290 32.682 1.00 0.02 ATOM 2237 CA GLU A 249 −6.581 −16.870 33.231 1.00 0.02 ATOM 2238 CB GLU A 249 −7.684 −17.300 32.272 1.00 0.71 ATOM 2239 CG GLU A 249 −7.580 −16.561 30.942 1.00 1.41 ATOM 2240 CD GLU A 249 −8.624 −17.099 29.972 1.00 1.73 ATOM 2241 OE1 GLU A 249 −9.040 −16.345 29.104 1.00 2.08 ATOM 2242 OE2 GLU A 249 −8.886 −18.292 30.041 1.00 2.14 ATOM 2243 C GLU A 249 −6.854 −17.455 34.619 1.00 0.02 ATOM 2244 O GLU A 249 −7.601 −16.869 35.408 1.00 0.02 ATOM 2245 N GLN A 250 −6.197 −18.560 34.932 1.00 0.01 ATOM 2247 CA GLN A 250 −6.243 −19.117 36.285 1.00 0.02 ATOM 2248 CB GLN A 250 −6.843 −20.516 36.228 1.00 0.06 ATOM 2249 CG GLN A 250 −8.295 −20.469 35.763 1.00 0.43 ATOM 2250 CD GLN A 250 −8.850 −21.881 35.628 1.00 0.27 ATOM 2251 OE1 GLN A 250 −10.050 −22.080 35.409 1.00 0.98 ATOM 2252 NE2 GLN A 250 −7.959 −22.849 35.739 1.00 1.30 ATOM 2255 C GLN A 250 −4.843 −19.169 36.893 1.00 0.01 ATOM 2256 O GLN A 250 −4.630 −19.809 37.928 1.00 0.01 ATOM 2257 N VAL A 251 −3.893 −18.537 36.222 1.00 0.01 ATOM 2259 CA VAL A 251 −2.492 −18.612 36.650 1.00 0.00 ATOM 2260 CB VAL A 251 −1.642 −19.165 35.509 1.00 0.50 ATOM 2261 CG1 VAL A 251 −0.215 −19.426 35.978 1.00 0.57 ATOM 2262 CG2 VAL A 251 −2.244 −20.448 34.946 1.00 0.85 ATOM 2263 C VAL A 251 −2.006 −17.224 37.055 1.00 0.01 ATOM 2264 O VAL A 251 −1.695 −16.371 36.209 1.00 0.01 ATOM 2265 N THR A 252 −1.927 −17.035 38.360 1.00 0.02 ATOM 2267 CA THR A 252 −1.641 −15.716 38.930 1.00 0.01 ATOM 2268 CB THR A 252 −2.806 −15.364 39.849 1.00 0.02 ATOM 2269 OG1 THR A 252 −2.848 −16.297 40.921 1.00 0.01 ATOM 2270 CG2 THR A 252 −4.150 −15.403 39.133 1.00 0.01 ATOM 2271 C THR A 252 −0.323 −15.655 39.714 1.00 0.02 ATOM 2272 O THR A 252 −0.042 −16.495 40.577 1.00 0.01 ATOM 2273 N VAL A 253 0.467 −14.638 39.408 1.00 0.02 ATOM 2275 CA VAL A 253 1.739 −14.387 40.116 1.00 0.00 ATOM 2276 CB VAL A 253 2.866 −14.411 39.084 1.00 0.01 ATOM 2277 CG1 VAL A 253 4.217 −14.060 39.701 1.00 0.03 ATOM 2278 CG2 VAL A 253 2.941 −15.761 38.378 1.00 0.02 ATOM 2279 C VAL A 253 1.714 −13.024 40.816 1.00 0.01 ATOM 2280 O VAL A 253 1.586 −11.997 40.144 1.00 0.02 ATOM 2281 N GLN A 254 1.809 −12.998 42.138 1.00 0.03 ATOM 2283 CA GLN A 254 1.732 −11.706 42.847 1.00 0.02 ATOM 2284 CB GLN A 254 1.677 −11.901 44.354 1.00 0.41 ATOM 2285 CG GLN A 254 0.309 −12.401 44.789 1.00 0.84 ATOM 2286 CD GLN A 254 0.199 −12.319 46.305 1.00 0.68 ATOM 2287 OE1 GLN A 254 0.343 −13.328 47.005 1.00 0.68 ATOM 2288 NE2 GLN A 254 −0.051 −11.115 46.791 1.00 1.21 ATOM 2291 C GLN A 254 2.892 −10.780 42.504 1.00 0.02 ATOM 2292 O GLN A 254 4.053 −11.191 42.424 1.00 0.03 ATOM 2293 N VAL A 255 2.537 −9.531 42.253 1.00 0.02 ATOM 2295 CA VAL A 255 3.523 −8.525 41.852 1.00 0.02 ATOM 2296 CB VAL A 255 3.330 −8.225 40.364 1.00 0.02 ATOM 2297 CG1 VAL A 255 3.834 −9.361 39.482 1.00 0.02 ATOM 2298 CG2 VAL A 255 1.877 −7.899 40.047 1.00 0.00 ATOM 2299 C VAL A 255 3.387 −7.241 42.668 1.00 0.02 ATOM 2300 O VAL A 255 2.295 −6.890 43.135 1.00 0.02 ATOM 2301 N PRO A 256 4.516 −6.581 42.876 1.00 0.02 ATOM 2302 CA PRO A 256 4.504 −5.197 43.353 1.00 0.02 ATOM 2303 CB PRO A 256 5.943 −4.796 43.459 1.00 0.02 ATOM 2304 CG PRO A 256 6.815 −5.925 42.928 1.00 0.00 ATOM 2305 CD PRO A 256 5.862 −7.035 42.515 1.00 0.02 ATOM 2306 C PRO A 256 3.748 −4.321 42.364 1.00 0.01 ATOM 2307 O PRO A 256 3.983 −4.375 41.151 1.00 0.01 ATOM 2308 N MET A 257 2.817 −3.551 42.888 1.00 0.02 ATOM 2310 CA MET A 257 1.925 −2.748 42.055 1.00 0.02 ATOM 2311 CB MET A 257 0.534 −3.335 42.232 1.00 0.01 ATOM 2312 CG MET A 257 −0.443 −2.884 41.158 1.00 0.02 ATOM 2313 SD MET A 257 −0.238 −3.681 39.550 1.00 0.02 ATOM 2314 CE MET A 257 −0.635 −5.384 39.996 1.00 0.02 ATOM 2315 C MET A 257 1.902 −1.296 42.515 1.00 0.00 ATOM 2316 O MET A 257 1.417 −0.989 43.610 1.00 0.02 ATOM 2317 N MET A 258 2.382 −0.413 41.659 1.00 0.01 ATOM 2319 CA MET A 258 2.388 1.020 41.954 1.00 0.02 ATOM 2320 CB MET A 258 3.323 1.701 40.968 1.00 0.01 ATOM 2321 CG MET A 258 4.699 1.054 40.968 1.00 0.01 ATOM 2322 SD MET A 258 5.891 1.774 39.818 1.00 0.01 ATOM 2323 CE MET A 258 4.983 1.573 38.271 1.00 0.00 ATOM 2324 C MET A 258 0.990 1.600 41.788 1.00 0.02 ATOM 2325 O MET A 258 0.451 1.661 40.674 1.00 0.02 ATOM 2326 N HIS A 259 0.397 1.948 42.917 1.00 0.01 ATOM 2328 CA HIS A 259 −0.940 2.541 42.928 1.00 0.01 ATOM 2329 CB HIS A 259 −1.625 2.053 44.202 1.00 0.78 ATOM 2330 CG HIS A 259 −3.078 2.437 44.412 1.00 1.36 ATOM 2331 ND1 HIS A 259 −3.735 2.391 45.585 1.00 2.20 ATOM 2333 CE1 HIS A 259 −5.007 2.802 45.401 1.00 2.85 ATOM 2334 NE2 HIS A 259 −5.158 3.092 44.089 1.00 2.78 ATOM 2335 CD2 HIS A 259 −3.981 2.861 43.465 1.00 2.13 ATOM 2336 C HIS A 259 −0.868 4.069 42.871 1.00 0.02 ATOM 2337 O HIS A 259 −0.052 4.713 43.548 1.00 0.02 ATOM 2338 N GLN A 260 −1.628 4.609 41.932 1.00 0.02 ATOM 2340 CA GLN A 260 −1.776 6.062 41.789 1.00 0.01 ATOM 2341 CB GLN A 260 −0.553 6.614 41.061 1.00 1.24 ATOM 2342 CG GLN A 260 −0.607 8.123 40.842 1.00 1.25 ATOM 2343 CD GLN A 260 −0.738 8.894 42.150 1.00 2.10 ATOM 2344 OE1 GLN A 260 −1.835 8.996 42.716 1.00 2.67 ATOM 2345 NE2 GLN A 260 0.355 9.512 42.553 1.00 2.69 ATOM 2348 C GLN A 260 −3.068 6.411 41.041 1.00 0.00 ATOM 2349 O GLN A 260 −3.191 6.194 39.829 1.00 0.01 ATOM 2350 N LYS A 261 −4.002 7.002 41.764 1.00 0.02 ATOM 2352 CA LYS A 261 −5.294 7.379 41.178 1.00 0.02 ATOM 2353 CB LYS A 261 −6.387 7.054 42.190 1.00 0.88 ATOM 2354 CG LYS A 261 −7.778 7.356 41.645 1.00 1.64 ATOM 2355 CD LYS A 261 −8.854 6.981 42.655 1.00 1.70 ATOM 2356 CE LYS A 261 −8.691 7.763 43.953 1.00 2.39 ATOM 2357 NZ LYS A 261 −9.741 7.398 44.917 1.00 2.92 ATOM 2358 C LYS A 261 −5.314 8.869 40.840 1.00 0.01 ATOM 2359 O LYS A 261 −5.677 9.699 41.681 1.00 0.01 ATOM 2360 N GLU A 262 −4.938 9.203 39.614 1.00 0.01 ATOM 2362 CA GLU A 262 −4.815 10.622 39.243 1.00 0.02 ATOM 2363 CB GLU A 262 −3.393 11.113 39.505 1.00 2.06 ATOM 2364 CG GLU A 262 −3.157 11.543 40.951 1.00 2.79 ATOM 2365 CD GLU A 262 −1.797 12.217 41.042 1.00 3.90 ATOM 2366 OE1 GLU A 262 −1.315 12.623 39.987 1.00 4.49 ATOM 2367 OE2 GLU A 262 −1.267 12.349 42.137 1.00 4.34 ATOM 2368 C GLU A 262 −5.178 10.992 37.802 1.00 0.02 ATOM 2369 O GLU A 262 −6.034 10.391 37.145 1.00 0.00 ATOM 2370 N GLN A 263 −4.375 11.934 37.326 1.00 0.01 ATOM 2372 CA GLN A 263 −4.570 12.772 36.122 1.00 0.00 ATOM 2373 CB GLN A 263 −3.615 13.938 36.349 1.00 1.50 ATOM 2374 CG GLN A 263 −2.219 13.357 36.596 1.00 2.17 ATOM 2375 CD GLN A 263 −1.135 14.425 36.683 1.00 3.16 ATOM 2376 OE1 GLN A 263 −0.815 15.081 35.688 1.00 3.64 ATOM 2377 NE2 GLN A 263 −0.508 14.507 37.845 1.00 3.93 ATOM 2380 C GLN A 263 −4.159 12.182 34.770 1.00 0.02 ATOM 2381 O GLN A 263 −3.864 12.951 33.851 1.00 0.01 ATOM 2382 N PHE A 264 −4.137 10.874 34.627 1.00 0.02 ATOM 2384 CA PHE A 264 −3.383 10.288 33.515 1.00 0.01 ATOM 2385 CB PHE A 264 −3.114 8.840 33.871 1.00 0.00 ATOM 2386 CG PHE A 264 −2.376 8.716 35.193 1.00 0.02 ATOM 2387 CD1 PHE A 264 −1.090 9.220 35.307 1.00 0.00 ATOM 2388 CE1 PHE A 264 −0.408 9.118 36.509 1.00 0.01 ATOM 2389 CZ PHE A 264 −1.017 8.520 37.601 1.00 0.00 ATOM 2390 CE2 PHE A 264 −2.311 8.034 37.494 1.00 0.01 ATOM 2391 CD2 PHE A 264 −2.995 8.137 36.292 1.00 0.02 ATOM 2392 C PHE A 264 −4.063 10.395 32.156 1.00 0.02 ATOM 2393 O PHE A 264 −5.282 10.242 32.023 1.00 0.00 ATOM 2394 N ALA A 265 −3.256 10.726 31.160 1.00 0.02 ATOM 2396 CA ALA A 265 −3.737 10.764 29.778 1.00 0.01 ATOM 2397 CB ALA A 265 −2.667 11.385 28.888 1.00 0.49 ATOM 2398 C ALA A 265 −4.049 9.342 29.339 1.00 0.01 ATOM 2399 O ALA A 265 −3.198 8.446 29.443 1.00 0.01 ATOM 2400 N PHE A 266 −5.265 9.141 28.868 1.00 0.01 ATOM 2402 CA PHE A 266 −5.734 7.775 28.640 1.00 0.01 ATOM 2403 CB PHE A 266 −6.156 7.234 30.003 1.00 1.51 ATOM 2404 CG PHE A 266 −6.592 5.776 30.027 1.00 1.98 ATOM 2405 CD1 PHE A 266 −5.645 4.769 29.916 1.00 2.64 ATOM 2406 CE1 PHE A 266 −6.042 3.441 29.929 1.00 3.43 ATOM 2407 CZ PHE A 266 −7.386 3.122 30.058 1.00 3.64 ATOM 2408 CE2 PHE A 266 −8.332 4.128 30.183 1.00 3.15 ATOM 2409 CD2 PHE A 266 −7.933 5.455 30.172 1.00 2.32 ATOM 2410 C PHE A 266 −6.904 7.704 27.665 1.00 0.01 ATOM 2411 O PHE A 266 −7.866 8.475 27.754 1.00 0.01 ATOM 2412 N GLY A 267 −6.821 6.749 26.755 1.00 0.01 ATOM 2414 CA GLY A 267 −7.928 6.507 25.824 1.00 0.00 ATOM 2415 C GLY A 267 −8.132 5.019 25.554 1.00 0.01 ATOM 2416 O GLY A 267 −7.181 4.231 25.560 1.00 0.01 ATOM 2417 N VAL A 268 −9.381 4.625 25.392 1.00 0.00 ATOM 2419 CA VAL A 268 −9.668 3.233 25.027 1.00 0.01 ATOM 2420 CB VAL A 268 −10.971 2.778 25.676 1.00 0.65 ATOM 2421 CG1 VAL A 268 −11.318 1.348 25.270 1.00 0.75 ATOM 2422 CG2 VAL A 268 −10.887 2.887 27.194 1.00 0.82 ATOM 2423 C VAL A 268 −9.748 3.112 23.508 1.00 0.01 ATOM 2424 O VAL A 268 −10.726 3.521 22.871 1.00 0.01 ATOM 2425 N ASP A 269 −8.692 2.563 22.940 1.00 0.01 ATOM 2427 CA ASP A 269 −8.605 2.409 21.493 1.00 0.00 ATOM 2428 CB ASP A 269 −7.123 2.419 21.123 1.00 0.44 ATOM 2429 CG ASP A 269 −6.905 2.536 19.616 1.00 0.29 ATOM 2430 OD1 ASP A 269 −6.024 3.277 19.222 1.00 0.56 ATOM 2431 OD2 ASP A 269 −7.593 1.833 18.883 1.00 0.37 ATOM 2432 C ASP A 269 −9.270 1.096 21.091 1.00 0.01 ATOM 2433 O ASP A 269 −8.686 0.017 21.240 1.00 0.01 ATOM 2434 N THR A 270 −10.371 1.211 20.369 1.00 0.00 ATOM 2436 CA THR A 270 −11.141 0.021 19.977 1.00 0.01 ATOM 2437 CB THR A 270 −12.550 0.481 19.617 1.00 0.50 ATOM 2438 OG1 THR A 270 −13.085 1.154 20.750 1.00 0.86 ATOM 2439 CG2 THR A 270 −13.474 −0.684 19.282 1.00 1.61 ATOM 2440 C THR A 270 −10.521 −0.760 18.810 1.00 0.00 ATOM 2441 O THR A 270 −10.683 −1.983 18.756 1.00 0.01 ATOM 2442 N GLU A 271 −9.583 −0.147 18.105 1.00 0.01 ATOM 2444 CA GLU A 271 −8.892 −0.832 17.005 1.00 0.00 ATOM 2445 CB GLU A 271 −8.414 0.220 16.012 1.00 0.34 ATOM 2446 CG GLU A 271 −9.585 1.034 15.473 1.00 0.93 ATOM 2447 CD GLU A 271 −9.085 2.127 14.536 1.00 0.82 ATOM 2448 OE1 GLU A 271 −8.023 1.944 13.958 1.00 0.84 ATOM 2449 OE2 GLU A 271 −9.733 3.165 14.488 1.00 1.00 ATOM 2450 C GLU A 271 −7.696 −1.638 17.517 1.00 0.01 ATOM 2451 O GLU A 271 −7.262 −2.594 16.869 1.00 0.01 ATOM 2452 N LEU A 272 −7.251 −1.310 18.721 1.00 0.00 ATOM 2454 CA LEU A 272 −6.228 −2.101 19.418 1.00 0.01 ATOM 2455 CB LEU A 272 −5.293 −1.154 20.163 1.00 0.01 ATOM 2456 CG LEU A 272 −4.450 −0.309 19.215 1.00 0.00 ATOM 2457 CD1 LEU A 272 −3.644 0.734 19.982 1.00 0.00 ATOM 2458 CD2 LEU A 272 −3.523 −1.188 18.386 1.00 0.01 ATOM 2459 C LEU A 272 −6.896 −3.040 20.418 1.00 0.01 ATOM 2460 O LEU A 272 −6.260 −3.935 20.987 1.00 0.00 ATOM 2461 N ASN A 273 −8.204 −2.877 20.518 1.00 0.01 ATOM 2463 CA ASN A 273 −9.056 −3.573 21.475 1.00 0.01 ATOM 2464 CB ASN A 273 −9.106 −5.052 21.124 1.00 0.59 ATOM 2465 CG ASN A 273 −10.529 −5.554 21.328 1.00 1.61 ATOM 2466 OD1 ASN A 273 −11.218 −5.159 22.278 1.00 2.19 ATOM 2467 ND2 ASN A 273 −10.962 −6.398 20.411 1.00 2.30 ATOM 2470 C ASN A 273 −8.580 −3.367 22.908 1.00 0.01 ATOM 2471 O ASN A 273 −7.951 −4.275 23.470 1.00 0.01 ATOM 2472 N CYS A 274 −8.633 −2.102 23.321 1.00 0.01 ATOM 2474 CA CYS A 274 −8.541 −1.636 24.728 1.00 0.00 ATOM 2475 CB CYS A 274 −8.367 −2.729 25.774 1.00 0.97 ATOM 2476 SG CYS A 274 −9.906 −3.343 26.496 1.00 2.23 ATOM 2477 C CYS A 274 −7.560 −0.496 25.011 1.00 0.01 ATOM 2478 O CYS A 274 −7.305 0.404 24.202 1.00 0.01 ATOM 2479 N PHE A 275 −7.079 −0.543 26.240 1.00 0.01 ATOM 2481 CA PHE A 275 −6.541 0.622 26.959 1.00 0.01 ATOM 2482 CB PHE A 275 −6.426 0.196 28.420 1.00 0.85 ATOM 2483 CG PHE A 275 −7.696 −0.445 28.981 1.00 1.11 ATOM 2484 CD1 PHE A 275 −8.913 0.223 28.910 1.00 1.88 ATOM 2485 CE1 PHE A 275 −10.064 −0.374 29.406 1.00 2.82 ATOM 2486 CZ PHE A 275 −9.999 −1.637 29.980 1.00 2.95 ATOM 2487 CE2 PHE A 275 −8.782 −2.300 30.062 1.00 2.21 ATOM 2488 CD2 PHE A 275 −7.631 −1.703 29.565 1.00 1.32 ATOM 2489 C PHE A 275 −5.181 1.126 26.485 1.00 0.01 ATOM 2490 O PHE A 275 −4.230 0.355 26.329 1.00 0.01 ATOM 2491 N VAL A 276 −5.105 2.430 26.274 1.00 0.01 ATOM 2493 CA VAL A 276 −3.837 3.106 25.971 1.00 0.00 ATOM 2494 CB VAL A 276 −3.917 3.722 24.575 1.00 0.00 ATOM 2495 CG1 VAL A 276 −2.630 4.465 24.228 1.00 0.00 ATOM 2496 CG2 VAL A 276 −4.222 2.669 23.515 1.00 0.01 ATOM 2497 C VAL A 276 −3.557 4.212 26.993 1.00 0.01 ATOM 2498 O VAL A 276 −4.204 5.270 26.990 1.00 0.01 ATOM 2499 N LEU A 277 −2.644 3.919 27.905 1.00 0.00 ATOM 2501 CA LEU A 277 −2.197 4.899 28.908 1.00 0.00 ATOM 2502 CB LEU A 277 −1.933 4.135 30.206 1.00 0.01 ATOM 2503 CG LEU A 277 −1.598 5.042 31.386 1.00 0.01 ATOM 2504 CD1 LEU A 277 −2.724 6.031 31.654 1.00 0.01 ATOM 2505 CD2 LEU A 277 −1.310 4.224 32.638 1.00 0.02 ATOM 2506 C LEU A 277 −0.921 5.591 28.423 1.00 0.01 ATOM 2507 O LEU A 277 −0.067 4.944 27.808 1.00 0.01 ATOM 2508 N GLN A 278 −0.822 6.894 28.624 1.00 0.01 ATOM 2510 CA GLN A 278 0.382 7.618 28.196 1.00 0.02 ATOM 2511 CB GLN A 278 0.085 8.219 26.831 1.00 0.54 ATOM 2512 CG GLN A 278 −1.353 8.713 26.749 1.00 0.92 ATOM 2513 CD GLN A 278 −1.695 9.059 25.309 1.00 0.81 ATOM 2514 OE1 GLN A 278 −1.901 8.168 24.475 1.00 1.23 ATOM 2515 NE2 GLN A 278 −1.791 10.347 25.047 1.00 1.11 ATOM 2518 C GLN A 278 0.855 8.675 29.201 1.00 0.01 ATOM 2519 O GLN A 278 0.274 9.761 29.337 1.00 0.00 ATOM 2520 N MET A 279 1.957 8.353 29.860 1.00 0.01 ATOM 2522 CA MET A 279 2.542 9.250 30.873 1.00 0.00 ATOM 2523 CB MET A 279 2.636 8.492 32.190 1.00 0.02 ATOM 2524 CG MET A 279 1.261 8.070 32.692 1.00 0.02 ATOM 2525 SD MET A 279 1.280 7.035 34.170 1.00 0.02 ATOM 2526 CE MET A 279 2.174 5.622 33.495 1.00 0.01 ATOM 2527 C MET A 279 3.937 9.737 30.481 1.00 0.01 ATOM 2528 O MET A 279 4.822 8.939 30.155 1.00 0.01 ATOM 2529 N ASP A 280 4.143 11.040 30.555 1.00 0.02 ATOM 2531 CA ASP A 280 5.448 11.608 30.190 1.00 0.01 ATOM 2532 CB ASP A 280 5.233 13.052 29.725 1.00 0.60 ATOM 2533 CG ASP A 280 6.513 13.690 29.173 1.00 1.52 ATOM 2534 OD1 ASP A 280 7.222 13.007 28.441 1.00 2.18 ATOM 2535 OD2 ASP A 280 6.670 14.888 29.350 1.00 1.96 ATOM 2536 C ASP A 280 6.442 11.557 31.355 1.00 0.01 ATOM 2537 O ASP A 280 6.096 11.768 32.529 1.00 0.00 ATOM 2538 N TYR A 281 7.657 11.163 31.014 1.00 0.01 ATOM 2540 CA TYR A 281 8.799 11.244 31.924 1.00 0.01 ATOM 2541 CB TYR A 281 9.948 10.412 31.369 1.00 0.01 ATOM 2542 CG TYR A 281 9.693 8.913 31.359 1.00 0.01 ATOM 2543 CD1 TYR A 281 9.194 8.289 32.494 1.00 0.00 ATOM 2544 CE1 TYR A 281 8.969 6.921 32.489 1.00 0.02 ATOM 2545 CZ TYR A 281 9.245 6.179 31.350 1.00 0.01 ATOM 2546 OH TYR A 281 9.043 4.816 31.357 1.00 0.00 ATOM 2547 CE2 TYR A 281 9.745 6.799 30.214 1.00 0.01 ATOM 2548 CD2 TYR A 281 9.972 8.168 30.220 1.00 0.00 ATOM 2549 C TYR A 281 9.255 12.687 32.035 1.00 0.01 ATOM 2550 O TYR A 281 8.700 13.569 31.367 1.00 0.02 ATOM 2551 N LYS A 282 10.160 12.940 32.962 1.00 0.02 ATOM 2553 CA LYS A 282 10.701 14.291 33.131 1.00 0.01 ATOM 2554 CB LYS A 282 11.492 14.351 34.433 1.00 0.41 ATOM 2555 CG LYS A 282 10.586 14.149 35.641 1.00 0.84 ATOM 2556 CD LYS A 282 11.364 14.264 36.948 1.00 1.00 ATOM 2557 CE LYS A 282 10.439 14.165 38.157 1.00 1.86 ATOM 2558 NZ LYS A 282 11.195 14.269 39.415 1.00 2.31 ATOM 2559 C LYS A 282 11.602 14.667 31.963 1.00 0.01 ATOM 2560 O LYS A 282 12.704 14.130 31.821 1.00 0.01 ATOM 2561 N GLY A 283 11.108 15.543 31.106 1.00 0.01 ATOM 2563 CA GLY A 283 11.923 16.019 29.986 1.00 0.01 ATOM 2564 C GLY A 283 11.242 15.903 28.625 1.00 0.01 ATOM 2565 O GLY A 283 10.740 16.897 28.090 1.00 0.01 ATOM 2566 N ASP A 284 11.319 14.720 28.034 1.00 0.02 ATOM 2568 CA ASP A 284 10.847 14.535 26.654 1.00 0.01 ATOM 2569 CB ASP A 284 11.811 15.258 25.704 1.00 0.72 ATOM 2570 CG ASP A 284 13.267 14.827 25.890 1.00 0.83 ATOM 2571 OD1 ASP A 284 13.687 13.930 25.169 1.00 1.50 ATOM 2572 OD2 ASP A 284 13.942 15.402 26.734 1.00 1.34 ATOM 2573 C ASP A 284 10.690 13.068 26.243 1.00 0.01 ATOM 2574 O ASP A 284 11.207 12.656 25.195 1.00 0.01 ATOM 2575 N ALA A 285 9.862 12.330 26.963 1.00 0.01 ATOM 2577 CA ALA A 285 9.665 10.911 26.629 1.00 0.00 ATOM 2578 CB ALA A 285 10.844 10.098 27.153 1.00 0.02 ATOM 2579 C ALA A 285 8.362 10.354 27.198 1.00 0.01 ATOM 2580 O ALA A 285 8.231 10.161 28.413 1.00 0.00 ATOM 2581 N VAL A 286 7.444 10.020 26.310 1.00 0.01 ATOM 2583 CA VAL A 286 6.142 9.503 26.737 1.00 0.01 ATOM 2584 CB VAL A 286 5.077 9.989 25.761 1.00 0.25 ATOM 2585 CG1 VAL A 286 3.682 9.553 26.198 1.00 0.48 ATOM 2586 CG2 VAL A 286 5.129 11.505 25.621 1.00 0.75 ATOM 2587 C VAL A 286 6.148 7.978 26.791 1.00 0.01 ATOM 2588 O VAL A 286 6.418 7.298 25.793 1.00 0.00 ATOM 2589 N ALA A 287 5.878 7.456 27.974 1.00 0.01 ATOM 2591 CA ALA A 287 5.757 6.012 28.160 1.00 0.01 ATOM 2592 CB ALA A 287 6.195 5.658 29.573 1.00 0.00 ATOM 2593 C ALA A 287 4.316 5.565 27.945 1.00 0.01 ATOM 2594 O ALA A 287 3.404 5.909 28.713 1.00 0.00 ATOM 2595 N PHE A 288 4.123 4.857 26.849 1.00 0.01 ATOM 2597 CA PHE A 288 2.825 4.271 26.525 1.00 0.01 ATOM 2598 CB PHE A 288 2.663 4.190 25.010 1.00 0.48 ATOM 2599 CG PHE A 288 2.617 5.528 24.268 1.00 0.62 ATOM 2600 CD1 PHE A 288 1.409 6.199 24.136 1.00 1.03 ATOM 2601 CE1 PHE A 288 1.355 7.409 23.454 1.00 1.26 ATOM 2602 CZ PHE A 288 2.509 7.946 22.898 1.00 1.09 ATOM 2603 CE2 PHE A 288 3.716 7.272 23.024 1.00 0.95 ATOM 2604 CD2 PHE A 288 3.769 6.064 23.705 1.00 0.77 ATOM 2605 C PHE A 288 2.695 2.876 27.131 1.00 0.01 ATOM 2606 O PHE A 288 3.632 2.066 27.116 1.00 0.01 ATOM 2607 N PHE A 289 1.533 2.653 27.717 1.00 0.00 ATOM 2609 CA PHE A 289 1.172 1.371 28.329 1.00 0.02 ATOM 2610 CB PHE A 289 1.036 1.550 29.837 1.00 0.02 ATOM 2611 CG PHE A 289 2.351 1.838 30.553 1.00 0.02 ATOM 2612 CD1 PHE A 289 3.146 0.780 30.970 1.00 0.01 ATOM 2613 CE1 PHE A 289 4.347 1.026 31.621 1.00 0.01 ATOM 2614 CZ PHE A 289 4.754 2.331 31.853 1.00 0.00 ATOM 2615 CE2 PHE A 289 3.958 3.391 31.439 1.00 0.02 ATOM 2616 CD2 PHE A 289 2.755 3.145 30.793 1.00 0.01 ATOM 2617 C PHE A 289 −0.146 0.882 27.747 1.00 0.01 ATOM 2618 O PHE A 289 −1.234 1.344 28.121 1.00 0.00 ATOM 2619 N VAL A 290 −0.030 −0.050 26.819 1.00 0.01 ATOM 2621 CA VAL A 290 −1.199 −0.530 26.087 1.00 0.01 ATOM 2622 CB VAL A 290 −0.838 −0.588 24.608 1.00 0.18 ATOM 2623 CG1 VAL A 290 −2.103 −0.578 23.760 1.00 0.27 ATOM 2624 CG2 VAL A 290 0.031 0.603 24.221 1.00 0.21 ATOM 2625 C VAL A 290 −1.639 −1.905 26.591 1.00 0.01 ATOM 2626 O VAL A 290 −0.972 −2.922 26.362 1.00 0.01 ATOM 2627 N LEU A 291 −2.783 −1.917 27.250 1.00 0.00 ATOM 2629 CA LEU A 291 −3.341 −3.146 27.826 1.00 0.01 ATOM 2630 CB LEU A 291 −3.874 −2.824 29.219 1.00 0.01 ATOM 2631 CG LEU A 291 −4.424 −4.056 29.932 1.00 0.01 ATOM 2632 CD1 LEU A 291 −3.342 −5.113 30.113 1.00 0.02 ATOM 2633 CD2 LEU A 291 −5.021 −3.677 31.281 1.00 0.01 ATOM 2634 C LEU A 291 −4.474 −3.678 26.953 1.00 0.01 ATOM 2635 O LEU A 291 −5.575 −3.111 26.930 1.00 0.00 ATOM 2636 N PRO A 292 −4.194 −4.771 26.261 1.00 0.01 ATOM 2637 CA PRO A 292 −5.169 −5.403 25.370 1.00 0.01 ATOM 2638 CB PRO A 292 −4.388 −6.418 24.595 1.00 0.01 ATOM 2639 CG PRO A 292 −2.992 −6.534 25.186 1.00 0.01 ATOM 2640 CD PRO A 292 −2.916 −5.489 26.284 1.00 0.01 ATOM 2641 C PRO A 292 −6.287 −6.090 26.139 1.00 0.01 ATOM 2642 O PRO A 292 −6.165 −6.362 27.340 1.00 0.02 ATOM 2643 N SER A 293 −7.386 −6.324 25.445 1.00 0.01 ATOM 2645 CA SER A 293 −8.472 −7.139 26.000 1.00 0.01 ATOM 2646 CB SER A 293 −9.636 −7.190 25.017 1.00 1.05 ATOM 2647 OG SER A 293 −10.092 −5.868 24.781 1.00 1.03 ATOM 2648 C SER A 293 −7.975 −8.559 26.229 1.00 0.01 ATOM 2649 O SER A 293 −7.012 −8.999 25.587 1.00 0.01 ATOM 2650 N LYS A 294 −8.613 −9.258 27.152 1.00 0.00 ATOM 2652 CA LYS A 294 −8.253 −10.655 27.434 1.00 0.01 ATOM 2653 CB LYS A 294 −9.124 −11.140 28.587 1.00 0.54 ATOM 2654 CG LYS A 294 −8.797 −12.572 28.996 1.00 1.54 ATOM 2655 CD LYS A 294 −9.634 −12.990 30.198 1.00 2.50 ATOM 2656 CE LYS A 294 −9.385 −12.059 31.380 1.00 3.48 ATOM 2657 NZ LYS A 294 −10.205 −12.443 32.539 1.00 4.07 ATOM 2658 C LYS A 294 −8.468 −11.533 26.199 1.00 0.01 ATOM 2659 O LYS A 294 −9.591 −11.681 25.707 1.00 0.01 ATOM 2660 N GLY A 295 −7.360 −11.997 25.639 1.00 0.01 ATOM 2662 CA GLY A 295 −7.391 −12.817 24.421 1.00 0.01 ATOM 2663 C GLY A 295 −6.939 −12.038 23.182 1.00 0.01 ATOM 2664 O GLY A 295 −6.305 −12.600 22.281 1.00 0.01 ATOM 2665 N LYS A 296 −7.107 −10.726 23.232 1.00 0.01 ATOM 2667 CA LYS A 296 −6.846 −9.849 22.085 1.00 0.01 ATOM 2668 CB LYS A 296 −7.853 −8.710 22.110 1.00 0.01 ATOM 2669 CG LYS A 296 −9.257 −9.233 21.840 1.00 0.01 ATOM 2670 CD LYS A 296 −9.341 −9.847 20.447 1.00 0.01 ATOM 2671 CE LYS A 296 −10.746 −10.345 20.138 1.00 0.00 ATOM 2672 NZ LYS A 296 −10.815 −10.887 18.773 1.00 0.01 ATOM 2673 C LYS A 296 −5.433 −9.279 22.051 1.00 0.01 ATOM 2674 O LYS A 296 −5.175 −8.337 21.290 1.00 0.01 ATOM 2675 N MET A 297 −4.504 −9.942 22.720 1.00 0.01 ATOM 2677 CA MET A 297 −3.123 −9.456 22.805 1.00 0.00 ATOM 2678 CB MET A 297 −2.353 −10.395 23.722 1.00 0.99 ATOM 2679 CG MET A 297 −0.922 −9.920 23.947 1.00 1.52 ATOM 2680 SD MET A 297 0.150 −11.110 24.780 1.00 1.96 ATOM 2681 CE MET A 297 −0.977 −11.607 26.104 1.00 1.91 ATOM 2682 C MET A 297 −2.448 −9.451 21.439 1.00 0.01 ATOM 2683 O MET A 297 −1.946 −8.402 21.013 1.00 0.01 ATOM 2684 N ARG A 298 −2.725 −10.481 20.655 1.00 0.01 ATOM 2686 CA ARG A 298 −2.131 −10.573 19.323 1.00 0.01 ATOM 2687 CB ARG A 298 −2.184 −12.028 18.874 1.00 0.22 ATOM 2688 CG ARG A 298 −1.429 −12.245 17.567 1.00 0.84 ATOM 2689 CD ARG A 298 0.040 −11.853 17.695 1.00 1.21 ATOM 2690 NE ARG A 298 0.759 −12.104 16.436 1.00 1.81 ATOM 2691 CZ ARG A 298 1.716 −11.304 15.959 1.00 2.42 ATOM 2692 NH1 ARG A 298 2.319 −11.599 14.805 1.00 3.43 ATOM 2693 NH2 ARG A 298 2.068 −10.206 16.632 1.00 2.42 ATOM 2694 C ARG A 298 −2.852 −9.676 18.318 1.00 0.01 ATOM 2695 O ARG A 298 −2.171 −9.052 17.497 1.00 0.01 ATOM 2696 N GLN A 299 −4.099 −9.330 18.599 1.00 0.01 ATOM 2698 CA GLN A 299 −4.825 −8.428 17.700 1.00 0.01 ATOM 2699 CB GLN A 299 −6.322 −8.497 17.984 1.00 0.22 ATOM 2700 CG GLN A 299 −7.083 −7.552 17.056 1.00 1.14 ATOM 2701 CD GLN A 299 −8.588 −7.630 17.297 1.00 1.84 ATOM 2702 OE1 GLN A 299 −9.099 −7.198 18.338 1.00 2.37 ATOM 2703 NE2 GLN A 299 −9.284 −8.196 16.326 1.00 2.56 ATOM 2706 C GLN A 299 −4.333 −6.997 17.881 1.00 0.01 ATOM 2707 O GLN A 299 −4.008 −6.348 16.877 1.00 0.01 ATOM 2708 N LEU A 300 −3.960 −6.671 19.110 1.00 0.01 ATOM 2710 CA LEU A 300 −3.388 −5.360 19.417 1.00 0.01 ATOM 2711 CB LEU A 300 −3.364 −5.224 20.941 1.00 0.01 ATOM 2712 CG LEU A 300 −2.876 −3.865 21.444 1.00 0.01 ATOM 2713 CD1 LEU A 300 −3.619 −3.466 22.711 1.00 0.01 ATOM 2714 CD2 LEU A 300 −1.366 −3.814 21.673 1.00 0.01 ATOM 2715 C LEU A 300 −1.979 −5.241 18.852 1.00 0.01 ATOM 2716 O LEU A 300 −1.670 −4.242 18.188 1.00 0.00 ATOM 2717 N GLU A 301 −1.244 −6.342 18.871 1.00 0.01 ATOM 2719 CA GLU A 301 0.128 −6.330 18.360 1.00 0.01 ATOM 2720 CB GLU A 301 0.833 −7.573 18.881 1.00 0.00 ATOM 2721 CG GLU A 301 0.969 −7.496 20.397 1.00 0.01 ATOM 2722 CD GLU A 301 1.457 −8.820 20.975 1.00 0.01 ATOM 2723 OE1 GLU A 301 0.861 −9.838 20.645 1.00 0.01 ATOM 2724 OE2 GLU A 301 2.301 −8.771 21.857 1.00 0.01 ATOM 2725 C GLU A 301 0.183 −6.273 16.835 1.00 0.00 ATOM 2726 O GLU A 301 1.031 −5.554 16.300 1.00 0.01 ATOM 2727 N GLN A 302 −0.854 −6.767 16.178 1.00 0.00 ATOM 2729 CA GLN A 302 −0.954 −6.664 14.717 1.00 0.01 ATOM 2730 CB GLN A 302 −1.851 −7.793 14.233 1.00 0.25 ATOM 2731 CG GLN A 302 −1.306 −9.172 14.574 1.00 0.93 ATOM 2732 CD GLN A 302 −2.446 −10.171 14.418 1.00 1.50 ATOM 2733 OE1 GLN A 302 −2.265 −11.388 14.534 1.00 2.23 ATOM 2734 NE2 GLN A 302 −3.632 −9.625 14.210 1.00 1.68 ATOM 2737 C GLN A 302 −1.588 −5.351 14.249 1.00 0.01 ATOM 2738 O GLN A 302 −1.615 −5.082 13.043 1.00 0.00 ATOM 2739 N ALA A 303 −2.109 −4.560 15.174 1.00 0.00 ATOM 2741 CA ALA A 303 −2.790 −3.316 14.808 1.00 0.00 ATOM 2742 CB ALA A 303 −4.138 −3.269 15.518 1.00 0.26 ATOM 2743 C ALA A 303 −1.975 −2.063 15.131 1.00 0.00 ATOM 2744 O ALA A 303 −2.486 −0.941 15.004 1.00 0.00 ATOM 2745 N LEU A 304 −0.737 −2.251 15.561 1.00 0.01 ATOM 2747 CA LEU A 304 0.133 −1.114 15.905 1.00 0.01 ATOM 2748 CB LEU A 304 1.343 −1.654 16.656 1.00 0.01 ATOM 2749 CG LEU A 304 0.954 −2.357 17.949 1.00 0.01 ATOM 2750 CD1 LEU A 304 2.151 −3.094 18.536 1.00 0.00 ATOM 2751 CD2 LEU A 304 0.366 −1.376 18.959 1.00 0.01 ATOM 2752 C LEU A 304 0.640 −0.360 14.674 1.00 0.01 ATOM 2753 O LEU A 304 1.621 −0.768 14.045 1.00 0.00 ATOM 2754 N SER A 305 −0.014 0.741 14.349 1.00 0.00 ATOM 2756 CA SER A 305 0.463 1.577 13.243 1.00 0.01 ATOM 2757 CB SER A 305 −0.707 2.019 12.371 1.00 0.42 ATOM 2758 OG SER A 305 −1.347 3.114 13.014 1.00 0.47 ATOM 2759 C SER A 305 1.161 2.818 13.779 1.00 0.00 ATOM 2760 O SER A 305 0.774 3.360 14.821 1.00 0.01 ATOM 2761 N ALA A 306 2.015 3.393 12.949 1.00 0.00 ATOM 2763 CA ALA A 306 2.696 4.636 13.326 1.00 0.01 ATOM 2764 CB ALA A 306 3.900 4.838 12.412 1.00 0.26 ATOM 2765 C ALA A 306 1.768 5.852 13.243 1.00 0.00 ATOM 2766 O ALA A 306 1.963 6.820 13.988 1.00 0.00 ATOM 2767 N ARG A 307 0.647 5.696 12.554 1.00 0.00 ATOM 2769 CA ARG A 307 −0.332 6.779 12.479 1.00 0.01 ATOM 2770 CB ARG A 307 −1.239 6.557 11.278 1.00 0.19 ATOM 2771 CG ARG A 307 −1.769 7.885 10.745 1.00 1.11 ATOM 2772 CD ARG A 307 −3.277 7.865 10.521 1.00 1.47 ATOM 2773 NE ARG A 307 −3.994 7.930 11.805 1.00 2.53 ATOM 2774 CZ ARG A 307 −4.809 6.974 12.255 1.00 3.45 ATOM 2775 NH1 ARG A 307 −4.995 5.862 11.541 1.00 4.09 ATOM 2776 NH2 ARG A 307 −5.419 7.122 13.432 1.00 4.06 ATOM 2777 C ARG A 307 −1.176 6.837 13.753 1.00 0.01 ATOM 2778 O ARG A 307 −1.359 7.931 14.297 1.00 0.00 ATOM 2779 N THR A 308 −1.485 5.692 14.349 1.00 0.01 ATOM 2781 CA THR A 308 −2.165 5.723 15.652 1.00 0.00 ATOM 2782 CB THR A 308 −2.832 4.376 15.929 1.00 0.18 ATOM 2783 OG1 THR A 308 −1.837 3.361 15.955 1.00 0.24 ATOM 2784 CG2 THR A 308 −3.858 4.012 14.863 1.00 0.45 ATOM 2785 C THR A 308 −1.187 6.045 16.780 1.00 0.01 ATOM 2786 O THR A 308 −1.579 6.676 17.767 1.00 0.00 ATOM 2787 N LEU A 309 0.093 5.852 16.514 1.00 0.01 ATOM 2789 CA LEU A 309 1.135 6.202 17.481 1.00 0.01 ATOM 2790 CB LEU A 309 2.444 5.577 17.012 1.00 0.17 ATOM 2791 CG LEU A 309 2.809 4.260 17.697 1.00 0.39 ATOM 2792 CD1 LEU A 309 1.665 3.253 17.805 1.00 0.43 ATOM 2793 CD2 LEU A 309 4.007 3.630 17.001 1.00 0.47 ATOM 2794 C LEU A 309 1.304 7.714 17.567 1.00 0.01 ATOM 2795 O LEU A 309 1.259 8.269 18.673 1.00 0.01 ATOM 2796 N ILE A 310 1.224 8.387 16.428 1.00 0.01 ATOM 2798 CA ILE A 310 1.309 9.849 16.443 1.00 0.01 ATOM 2799 CB ILE A 310 1.872 10.353 15.112 1.00 0.24 ATOM 2800 CG2 ILE A 310 0.948 10.046 13.940 1.00 0.79 ATOM 2801 CG1 ILE A 310 2.160 11.849 15.168 1.00 1.18 ATOM 2802 CD1 ILE A 310 2.720 12.350 13.842 1.00 1.93 ATOM 2803 C ILE A 310 −0.043 10.487 16.783 1.00 0.01 ATOM 2804 O ILE A 310 −0.070 11.594 17.336 1.00 0.01 ATOM 2805 N LYS A 311 −1.107 9.702 16.703 1.00 0.01 ATOM 2807 CA LYS A 311 −2.419 10.150 17.164 1.00 0.01 ATOM 2808 CB LYS A 311 −3.464 9.156 16.677 1.00 0.01 ATOM 2809 CG LYS A 311 −4.850 9.489 17.209 1.00 0.01 ATOM 2810 CD LYS A 311 −5.842 8.389 16.858 1.00 0.01 ATOM 2811 CE LYS A 311 −7.228 8.691 17.414 1.00 0.00 ATOM 2812 NZ LYS A 311 −8.166 7.606 17.087 1.00 0.01 ATOM 2813 C LYS A 311 −2.452 10.227 18.686 1.00 0.01 ATOM 2814 O LYS A 311 −2.705 11.305 19.242 1.00 0.01 ATOM 2815 N TRP A 312 −1.911 9.206 19.329 1.00 0.01 ATOM 2817 CA TRP A 312 −1.874 9.211 20.789 1.00 0.01 ATOM 2818 CB TRP A 312 −1.702 7.784 21.291 1.00 0.26 ATOM 2819 CG TRP A 312 −2.941 6.939 21.080 1.00 0.93 ATOM 2820 CD1 TRP A 312 −3.058 5.824 20.281 1.00 2.03 ATOM 2821 NE1 TRP A 312 −4.334 5.374 20.351 1.00 2.63 ATOM 2823 CE2 TRP A 312 −5.073 6.138 21.177 1.00 1.99 ATOM 2824 CZ2 TRP A 312 −6.399 6.090 21.558 1.00 2.30 ATOM 2825 CH2 TRP A 312 −6.898 7.040 22.452 1.00 1.63 ATOM 2826 CZ3 TRP A 312 −6.068 8.027 22.958 1.00 0.98 ATOM 2827 CE3 TRP A 312 −4.730 8.082 22.576 1.00 0.71 ATOM 2828 CD2 TRP A 312 −4.230 7.143 21.684 1.00 0.94 ATOM 2829 C TRP A 312 −0.777 10.113 21.338 1.00 0.00 ATOM 2830 O TRP A 312 −1.025 10.794 22.338 1.00 0.01 ATOM 2831 N SER A 313 0.236 10.390 20.534 1.00 0.00 ATOM 2833 CA SER A 313 1.279 11.321 20.976 1.00 0.00 ATOM 2834 CB SER A 313 2.560 11.069 20.190 1.00 0.24 ATOM 2835 OG SER A 313 2.342 11.501 18.857 1.00 0.20 ATOM 2836 C SER A 313 0.882 12.796 20.830 1.00 0.01 ATOM 2837 O SER A 313 1.640 13.656 21.290 1.00 0.00 ATOM 2838 N HIS A 314 −0.245 13.105 20.200 1.00 0.01 ATOM 2840 CA HIS A 314 −0.728 14.488 20.258 1.00 0.01 ATOM 2841 CB HIS A 314 −1.012 15.063 18.869 1.00 0.13 ATOM 2842 CG HIS A 314 −2.195 14.499 18.103 1.00 0.18 ATOM 2843 ND1 HIS A 314 −2.128 13.688 17.033 1.00 0.25 ATOM 2845 CE1 HIS A 314 −3.375 13.404 16.607 1.00 0.30 ATOM 2846 NE2 HIS A 314 −4.242 14.049 17.420 1.00 0.28 ATOM 2847 CD2 HIS A 314 −3.530 14.737 18.340 1.00 0.22 ATOM 2848 C HIS A 314 −1.958 14.594 21.154 1.00 0.01 ATOM 2849 O HIS A 314 −2.450 15.700 21.411 1.00 0.01 ATOM 2850 N SER A 315 −2.462 13.456 21.603 1.00 0.01 ATOM 2852 CA SER A 315 −3.602 13.466 22.521 1.00 0.00 ATOM 2853 CB SER A 315 −4.209 12.075 22.626 1.00 1.14 ATOM 2854 OG SER A 315 −5.144 12.131 23.697 1.00 0.90 ATOM 2855 C SER A 315 −3.187 13.914 23.912 1.00 0.01 ATOM 2856 O SER A 315 −2.451 13.230 24.629 1.00 0.01 ATOM 2857 N LEU A 316 −3.759 15.029 24.321 1.00 0.00 ATOM 2859 CA LEU A 316 −3.489 15.563 25.654 1.00 0.01 ATOM 2860 CB LEU A 316 −3.311 17.071 25.509 1.00 0.88 ATOM 2861 CG LEU A 316 −2.537 17.664 26.682 1.00 2.18 ATOM 2862 CD1 LEU A 316 −1.203 16.939 26.876 1.00 2.44 ATOM 2863 CD2 LEU A 316 −2.336 19.167 26.493 1.00 3.16 ATOM 2864 C LEU A 316 −4.621 15.225 26.633 1.00 0.01 ATOM 2865 O LEU A 316 −4.649 15.755 27.750 1.00 0.01 ATOM 2866 N GLN A 317 −5.507 14.318 26.241 1.00 0.01 ATOM 2868 CA GLN A 317 −6.718 14.058 27.030 1.00 0.00 ATOM 2869 CB GLN A 317 −7.740 13.326 26.166 1.00 0.55 ATOM 2870 CG GLN A 317 −9.095 13.217 26.866 1.00 1.06 ATOM 2871 CD GLN A 317 −9.686 14.609 27.104 1.00 1.59 ATOM 2872 OE1 GLN A 317 −9.231 15.360 27.973 1.00 2.10 ATOM 2873 NE2 GLN A 317 −10.682 14.947 26.304 1.00 2.07 ATOM 2876 C GLN A 317 −6.428 13.248 28.288 1.00 0.01 ATOM 2877 O GLN A 317 −6.204 12.030 28.245 1.00 0.01 ATOM 2878 N LYS A 318 −6.423 13.963 29.397 1.00 0.01 ATOM 2880 CA LYS A 318 −6.161 13.376 30.708 1.00 0.00 ATOM 2881 CB LYS A 318 −5.293 14.346 31.495 1.00 0.64 ATOM 2882 CG LYS A 318 −3.944 14.570 30.825 1.00 1.38 ATOM 2883 CD LYS A 318 −3.065 15.477 31.674 1.00 1.55 ATOM 2884 CE LYS A 318 −1.700 15.689 31.033 1.00 2.51 ATOM 2885 NZ LYS A 318 −0.837 16.502 31.905 1.00 3.10 ATOM 2886 C LYS A 318 −7.452 13.143 31.474 1.00 0.02 ATOM 2887 O LYS A 318 −8.345 13.997 31.492 1.00 0.01 ATOM 2888 N ARG A 319 −7.552 11.986 32.098 1.00 0.02 ATOM 2890 CA ARG A 319 −8.712 11.728 32.945 1.00 0.01 ATOM 2891 CB ARG A 319 −9.810 11.008 32.171 1.00 0.01 ATOM 2892 CG ARG A 319 −9.397 9.697 31.517 1.00 0.00 ATOM 2893 CD ARG A 319 −10.610 9.150 30.774 1.00 0.00 ATOM 2894 NE ARG A 319 −10.350 7.878 30.088 1.00 0.02 ATOM 2895 CZ ARG A 319 −10.965 7.547 28.950 1.00 0.01 ATOM 2896 NH1 ARG A 319 −11.774 8.423 28.352 1.00 0.01 ATOM 2897 NH2 ARG A 319 −10.727 6.365 28.378 1.00 0.01 ATOM 2898 C ARG A 319 −8.346 10.999 34.231 1.00 0.02 ATOM 2899 O ARG A 319 −7.237 10.488 34.422 1.00 0.01 ATOM 2900 N TRP A 320 −9.294 11.068 35.145 1.00 0.02 ATOM 2902 CA TRP A 320 −9.134 10.543 36.499 1.00 0.01 ATOM 2903 CB TRP A 320 −10.232 11.177 37.346 1.00 2.46 ATOM 2904 CG TRP A 320 −10.407 12.649 37.031 1.00 3.22 ATOM 2905 CD1 TRP A 320 −9.571 13.677 37.406 1.00 4.15 ATOM 2906 NE1 TRP A 320 −10.048 14.835 36.887 1.00 4.95 ATOM 2908 CE2 TRP A 320 −11.170 14.620 36.167 1.00 4.71 ATOM 2909 CZ2 TRP A 320 −11.968 15.453 35.399 1.00 5.57 ATOM 2910 CH2 TRP A 320 −13.083 14.932 34.750 1.00 5.51 ATOM 2911 CZ3 TRP A 320 −13.393 13.580 34.861 1.00 4.74 ATOM 2912 CE3 TRP A 320 −12.587 12.736 35.613 1.00 3.74 ATOM 2913 CD2 TRP A 320 −11.471 13.253 36.260 1.00 3.68 ATOM 2914 C TRP A 320 −9.278 9.032 36.489 1.00 0.00 ATOM 2915 O TRP A 320 −10.354 8.506 36.190 1.00 0.00 ATOM 2916 N ILE A 321 −8.172 8.350 36.720 1.00 0.01 ATOM 2918 CA ILE A 321 −8.166 6.881 36.726 1.00 0.00 ATOM 2919 CB ILE A 321 −7.656 6.390 35.378 1.00 0.72 ATOM 2920 CG2 ILE A 321 −8.752 6.400 34.315 1.00 1.67 ATOM 2921 CG1 ILE A 321 −6.452 7.206 34.930 1.00 1.15 ATOM 2922 CD1 ILE A 321 −5.986 6.773 33.549 1.00 1.60 ATOM 2923 C ILE A 321 −7.317 6.306 37.857 1.00 0.02 ATOM 2924 O ILE A 321 −6.287 6.869 38.249 1.00 0.00 ATOM 2925 N GLU A 322 −7.732 5.140 38.323 1.00 0.01 ATOM 2927 CA GLU A 322 −7.086 4.466 39.461 1.00 0.02 ATOM 2928 CB GLU A 322 −8.198 3.733 40.205 1.00 0.75 ATOM 2929 CG GLU A 322 −7.735 3.098 41.508 1.00 1.32 ATOM 2930 CD GLU A 322 −8.891 2.309 42.108 1.00 1.58 ATOM 2931 OE1 GLU A 322 −8.813 1.994 43.287 1.00 1.93 ATOM 2932 OE2 GLU A 322 −9.859 2.086 41.392 1.00 1.79 ATOM 2933 C GLU A 322 −6.012 3.474 38.997 1.00 0.02 ATOM 2934 O GLU A 322 −6.201 2.254 39.077 1.00 0.02 ATOM 2935 N VAL A 323 −4.871 4.007 38.597 1.00 0.00 ATOM 2937 CA VAL A 323 −3.819 3.209 37.952 1.00 0.00 ATOM 2938 CB VAL A 323 −2.852 4.200 37.307 1.00 0.58 ATOM 2939 CG1 VAL A 323 −1.511 3.597 36.913 1.00 1.12 ATOM 2940 CG2 VAL A 323 −3.507 4.860 36.104 1.00 1.30 ATOM 2941 C VAL A 323 −3.095 2.236 38.885 1.00 0.02 ATOM 2942 O VAL A 323 −2.768 2.554 40.038 1.00 0.00 ATOM 2943 N PHE A 324 −2.997 1.007 38.396 1.00 0.02 ATOM 2945 CA PHE A 324 −2.221 −0.061 39.037 1.00 0.02 ATOM 2946 CB PHE A 324 −3.167 −1.200 39.409 1.00 0.70 ATOM 2947 CG PHE A 324 −4.225 −0.894 40.461 1.00 0.66 ATOM 2948 CD1 PHE A 324 −3.850 −0.696 41.783 1.00 1.33 ATOM 2949 CE1 PHE A 324 −4.815 −0.432 42.745 1.00 2.10 ATOM 2950 CZ PHE A 324 −6.155 −0.369 42.387 1.00 2.27 ATOM 2951 CE2 PHE A 324 −6.531 −0.571 41.065 1.00 1.95 ATOM 2952 CD2 PHE A 324 −5.566 −0.837 40.103 1.00 1.28 ATOM 2953 C PHE A 324 −1.190 −0.636 38.063 1.00 0.01 ATOM 2954 O PHE A 324 −1.516 −1.572 37.320 1.00 0.00 ATOM 2955 N ILE A 325 0.034 −0.128 38.092 1.00 0.01 ATOM 2957 CA ILE A 325 1.088 −0.642 37.188 1.00 0.02 ATOM 2958 CB ILE A 325 1.659 0.527 36.379 1.00 0.42 ATOM 2959 CG2 ILE A 325 2.869 0.119 35.543 1.00 0.61 ATOM 2960 CG1 ILE A 325 0.598 1.123 35.468 1.00 0.74 ATOM 2961 CD1 ILE A 325 1.195 2.203 34.575 1.00 1.12 ATOM 2962 C ILE A 325 2.209 −1.345 37.961 1.00 0.02 ATOM 2963 O ILE A 325 2.764 −0.770 38.901 1.00 0.01 ATOM 2964 N PRO A 326 2.521 −2.577 37.588 1.00 0.00 ATOM 2965 CA PRO A 326 3.556 −3.351 38.287 1.00 0.02 ATOM 2966 CB PRO A 326 3.580 −4.687 37.615 1.00 0.02 ATOM 2967 CG PRO A 326 2.546 −4.703 36.502 1.00 0.00 ATOM 2968 CD PRO A 326 1.890 −3.333 36.507 1.00 0.02 ATOM 2969 C PRO A 326 4.932 −2.683 38.260 1.00 0.00 ATOM 2970 O PRO A 326 5.296 −1.981 37.309 1.00 0.02 ATOM 2971 N ARG A 327 5.646 −2.858 39.358 1.00 0.01 ATOM 2973 CA ARG A 327 6.977 −2.267 39.528 1.00 0.02 ATOM 2974 CB ARG A 327 7.056 −1.674 40.929 1.00 0.43 ATOM 2975 CG ARG A 327 8.383 −0.959 41.131 1.00 1.22 ATOM 2976 CD ARG A 327 8.580 −0.486 42.561 1.00 1.09 ATOM 2977 NE ARG A 327 9.842 0.261 42.668 1.00 2.19 ATOM 2978 CZ ARG A 327 10.975 −0.253 43.151 1.00 2.68 ATOM 2979 NH1 ARG A 327 12.079 0.495 43.192 1.00 3.44 ATOM 2980 NH2 ARG A 327 11.008 −1.517 43.581 1.00 2.85 ATOM 2981 C ARG A 327 8.077 −3.316 39.398 1.00 0.02 ATOM 2982 O ARG A 327 8.186 −4.202 40.256 1.00 0.02 ATOM 2983 N PHE A 328 8.933 −3.157 38.399 1.00 0.02 ATOM 2985 CA PHE A 328 10.045 −4.102 38.206 1.00 0.01 ATOM 2986 CB PHE A 328 9.496 −5.484 37.861 1.00 1.21 ATOM 2987 CG PHE A 328 10.160 −6.623 38.635 1.00 2.16 ATOM 2988 CD1 PHE A 328 9.523 −7.158 39.747 1.00 3.00 ATOM 2989 CE1 PHE A 328 10.117 −8.194 40.457 1.00 3.93 ATOM 2990 CZ PHE A 328 11.348 −8.696 40.054 1.00 4.19 ATOM 2991 CE2 PHE A 328 11.984 −8.161 38.942 1.00 3.65 ATOM 2992 CD2 PHE A 328 11.390 −7.126 38.233 1.00 2.61 ATOM 2993 C PHE A 328 10.986 −3.651 37.091 1.00 0.01 ATOM 2994 O PHE A 328 10.583 −2.979 36.135 1.00 0.00 ATOM 2995 N SER A 329 12.254 −3.988 37.242 1.00 0.01 ATOM 2997 CA SER A 329 13.218 −3.718 36.176 1.00 0.01 ATOM 2998 CB SER A 329 14.586 −3.439 36.775 1.00 0.00 ATOM 2999 OG SER A 329 15.460 −3.178 35.685 1.00 0.01 ATOM 3000 C SER A 329 13.324 −4.900 35.215 1.00 0.02 ATOM 3001 O SER A 329 13.844 −5.964 35.567 1.00 0.02 ATOM 3002 N ILE A 330 12.857 −4.688 33.998 1.00 0.01 ATOM 3004 CA ILE A 330 12.930 −5.723 32.962 1.00 0.00 ATOM 3005 CB ILE A 330 11.662 −5.673 32.114 1.00 0.01 ATOM 3006 CG2 ILE A 330 10.451 −6.083 32.946 1.00 0.01 ATOM 3007 CG1 ILE A 330 11.456 −4.287 31.511 1.00 0.02 ATOM 3008 CD1 ILE A 330 10.201 −4.238 30.646 1.00 0.00 ATOM 3009 C ILE A 330 14.170 −5.524 32.092 1.00 0.01 ATOM 3010 O ILE A 330 14.637 −4.397 31.897 1.00 0.01 ATOM 3011 N SER A 331 14.740 −6.622 31.631 1.00 0.01 ATOM 3013 CA SER A 331 15.937 −6.517 30.790 1.00 0.00 ATOM 3014 CB SER A 331 17.164 −6.528 31.687 1.00 0.38 ATOM 3015 OG SER A 331 18.286 −6.315 30.846 1.00 0.92 ATOM 3016 C SER A 331 16.031 −7.657 29.782 1.00 0.00 ATOM 3017 O SER A 331 15.846 −8.829 30.131 1.00 0.01 ATOM 3018 N ALA A 332 16.368 −7.307 28.552 1.00 0.02 ATOM 3020 CA ALA A 332 16.466 −8.314 27.494 1.00 0.01 ATOM 3021 CB ALA A 332 15.182 −8.277 26.679 1.00 0.17 ATOM 3022 C ALA A 332 17.683 −8.130 26.585 1.00 0.01 ATOM 3023 O ALA A 332 17.896 −7.074 25.973 1.00 0.02 ATOM 3024 N SER A 333 18.492 −9.175 26.538 1.00 0.02 ATOM 3026 CA SER A 333 19.650 −9.212 25.635 1.00 0.02 ATOM 3027 CB SER A 333 20.804 −9.957 26.298 1.00 0.36 ATOM 3028 OG SER A 333 20.418 −11.313 26.472 1.00 1.04 ATOM 3029 C SER A 333 19.281 −9.904 24.325 1.00 0.02 ATOM 3030 O SER A 333 18.520 −10.878 24.311 1.00 0.01 ATOM 3031 N TYR A 334 19.810 −9.379 23.234 1.00 0.00 ATOM 3033 CA TYR A 334 19.513 −9.914 21.898 1.00 0.01 ATOM 3034 CB TYR A 334 18.510 −9.005 21.194 1.00 0.01 ATOM 3035 CG TYR A 334 17.169 −8.855 21.903 1.00 0.01 ATOM 3036 CD1 TYR A 334 16.270 −9.915 21.930 1.00 0.01 ATOM 3037 CE1 TYR A 334 15.052 −9.775 22.583 1.00 0.01 ATOM 3038 CZ TYR A 334 14.740 −8.574 23.205 1.00 0.00 ATOM 3039 OH TYR A 334 13.541 −8.429 23.867 1.00 0.01 ATOM 3040 CE2 TYR A 334 15.636 −7.516 23.178 1.00 0.01 ATOM 3041 CD2 TYR A 334 16.851 −7.657 22.527 1.00 0.01 ATOM 3042 C TYR A 334 20.767 −10.002 21.036 1.00 0.01 ATOM 3043 O TYR A 334 21.439 −8.995 20.779 1.00 0.01 ATOM 3044 N ASN A 335 21.063 −11.202 20.573 1.00 0.01 ATOM 3046 CA ASN A 335 22.186 −11.373 19.652 1.00 0.01 ATOM 3047 CB ASN A 335 22.629 −12.831 19.652 1.00 0.26 ATOM 3048 CG ASN A 335 23.835 −13.005 18.736 1.00 0.35 ATOM 3049 OD1 ASN A 335 23.683 −13.139 17.515 1.00 0.52 ATOM 3050 ND2 ASN A 335 25.016 −12.899 19.318 1.00 0.87 ATOM 3053 C ASN A 335 21.755 −10.940 18.254 1.00 0.01 ATOM 3054 O ASN A 335 21.077 −11.684 17.532 1.00 0.01 ATOM 3055 N LEU A 336 22.375 −9.868 17.792 1.00 0.01 ATOM 3057 CA LEU A 336 21.956 −9.223 16.544 1.00 0.01 ATOM 3058 CB LEU A 336 22.514 −7.805 16.520 1.00 0.01 ATOM 3059 CG LEU A 336 21.987 −6.958 17.671 1.00 0.01 ATOM 3060 CD1 LEU A 336 22.678 −5.599 17.697 1.00 0.01 ATOM 3061 CD2 LEU A 336 20.472 −6.796 17.582 1.00 0.01 ATOM 3062 C LEU A 336 22.463 −9.964 15.317 1.00 0.01 ATOM 3063 O LEU A 336 21.785 −9.955 14.284 1.00 0.01 ATOM 3064 N GLU A 337 23.419 −10.849 15.532 1.00 0.01 ATOM 3066 CA GLU A 337 24.019 −11.609 14.439 1.00 0.01 ATOM 3067 CB GLU A 337 25.387 −12.045 14.937 1.00 0.13 ATOM 3068 CG GLU A 337 26.202 −12.823 13.918 1.00 1.05 ATOM 3069 CD GLU A 337 27.589 −12.964 14.512 1.00 0.89 ATOM 3070 OE1 GLU A 337 28.180 −14.022 14.372 1.00 1.40 ATOM 3071 OE2 GLU A 337 27.896 −12.098 15.329 1.00 0.86 ATOM 3072 C GLU A 337 23.167 −12.815 14.048 1.00 0.01 ATOM 3073 O GLU A 337 23.318 −13.346 12.942 1.00 0.01 ATOM 3074 N THR A 338 22.229 −13.179 14.906 1.00 0.01 ATOM 3076 CA THR A 338 21.265 −14.229 14.568 1.00 0.01 ATOM 3077 CB THR A 338 21.139 −15.195 15.741 1.00 0.22 ATOM 3078 OG1 THR A 338 20.704 −14.465 16.881 1.00 0.83 ATOM 3079 CG2 THR A 338 22.473 −15.854 16.073 1.00 0.78 ATOM 3080 C THR A 338 19.892 −13.646 14.238 1.00 0.01 ATOM 3081 O THR A 338 19.056 −14.333 13.639 1.00 0.01 ATOM 3082 N ILE A 339 19.695 −12.372 14.539 1.00 0.01 ATOM 3084 CA ILE A 339 18.391 −11.748 14.284 1.00 0.01 ATOM 3085 CB ILE A 339 18.102 −10.748 15.398 1.00 0.19 ATOM 3086 CG2 ILE A 339 16.796 −10.018 15.118 1.00 0.33 ATOM 3087 CG1 ILE A 339 18.042 −11.425 16.765 1.00 0.43 ATOM 3088 CD1 ILE A 339 16.880 −12.407 16.865 1.00 0.67 ATOM 3089 C ILE A 339 18.363 −11.030 12.938 1.00 0.01 ATOM 3090 O ILE A 339 17.499 −11.303 12.093 1.00 0.01 ATOM 3091 N LEU A 340 19.419 −10.289 12.665 1.00 0.01 ATOM 3093 CA LEU A 340 19.487 −9.512 11.426 1.00 0.01 ATOM 3094 CB LEU A 340 20.616 −8.504 11.556 1.00 0.01 ATOM 3095 CG LEU A 340 20.352 −7.548 12.713 1.00 0.01 ATOM 3096 CD1 LEU A 340 21.572 −6.681 12.990 1.00 0.01 ATOM 3097 CD2 LEU A 340 19.114 −6.693 12.457 1.00 0.01 ATOM 3098 C LEU A 340 19.624 −10.296 10.102 1.00 0.01 ATOM 3099 O LEU A 340 19.003 −9.791 9.156 1.00 0.01 ATOM 3100 N PRO A 341 20.199 −11.500 9.987 1.00 0.01 ATOM 3101 CA PRO A 341 20.074 −12.210 8.702 1.00 0.01 ATOM 3102 CB PRO A 341 20.942 −13.425 8.813 1.00 0.39 ATOM 3103 CG PRO A 341 21.497 −13.522 10.219 1.00 0.63 ATOM 3104 CD PRO A 341 20.982 −12.297 10.948 1.00 0.66 ATOM 3105 C PRO A 341 18.642 −12.616 8.337 1.00 0.01 ATOM 3106 O PRO A 341 18.306 −12.529 7.150 1.00 0.01 ATOM 3107 N LYS A 342 17.752 −12.711 9.318 1.00 0.01 ATOM 3109 CA LYS A 342 16.348 −13.035 9.039 1.00 0.01 ATOM 3110 CB LYS A 342 15.712 −13.544 10.325 1.00 0.97 ATOM 3111 CG LYS A 342 16.524 −14.662 10.964 1.00 1.75 ATOM 3112 CD LYS A 342 15.847 −15.142 12.241 1.00 2.93 ATOM 3113 CE LYS A 342 15.599 −13.977 13.193 1.00 3.48 ATOM 3114 NZ LYS A 342 14.897 −14.426 14.405 1.00 3.77 ATOM 3115 C LYS A 342 15.574 −11.796 8.584 1.00 0.01 ATOM 3116 O LYS A 342 14.480 −11.904 8.018 1.00 0.01 ATOM 3117 N MET A 343 16.172 −10.634 8.797 1.00 0.01 ATOM 3119 CA MET A 343 15.594 −9.369 8.350 1.00 0.01 ATOM 3120 CB MET A 343 15.740 −8.343 9.469 1.00 1.42 ATOM 3121 CG MET A 343 15.088 −8.823 10.762 1.00 2.03 ATOM 3122 SD MET A 343 13.348 −9.309 10.639 1.00 1.82 ATOM 3123 CE MET A 343 12.684 −7.810 9.878 1.00 2.49 ATOM 3124 C MET A 343 16.269 −8.851 7.079 1.00 0.01 ATOM 3125 O MET A 343 15.957 −7.746 6.620 1.00 0.00 ATOM 3126 N GLY A 344 17.237 −9.591 6.563 1.00 0.01 ATOM 3128 CA GLY A 344 17.883 −9.196 5.306 1.00 0.01 ATOM 3129 C GLY A 344 19.370 −8.887 5.460 1.00 0.01 ATOM 3130 O GLY A 344 20.141 −9.041 4.504 1.00 0.01 ATOM 3131 N ILE A 345 19.760 −8.444 6.644 1.00 0.01 ATOM 3133 CA ILE A 345 21.156 −8.078 6.921 1.00 0.01 ATOM 3134 CB ILE A 345 21.159 −7.123 8.109 1.00 0.01 ATOM 3135 CG2 ILE A 345 22.529 −6.475 8.275 1.00 0.00 ATOM 3136 CG1 ILE A 345 20.086 −6.054 7.943 1.00 0.01 ATOM 3137 CD1 ILE A 345 20.083 −5.075 9.111 1.00 0.00 ATOM 3138 C ILE A 345 21.976 −9.320 7.271 1.00 0.00 ATOM 3139 O ILE A 345 22.186 −9.637 8.447 1.00 0.01 ATOM 3140 N GLN A 346 22.471 −9.992 6.247 1.00 0.00 ATOM 3142 CA GLN A 346 23.161 −11.269 6.443 1.00 0.01 ATOM 3143 CB GLN A 346 22.500 −12.262 5.500 1.00 1.21 ATOM 3144 CG GLN A 346 23.176 −13.625 5.548 1.00 1.87 ATOM 3145 CD GLN A 346 22.735 −14.438 4.339 1.00 2.20 ATOM 3146 OE1 GLN A 346 21.841 −15.289 4.431 1.00 2.70 ATOM 3147 NE2 GLN A 346 23.345 −14.135 3.205 1.00 2.46 ATOM 3150 C GLN A 346 24.640 −11.201 6.090 1.00 0.00 ATOM 3151 O GLN A 346 25.502 −11.688 6.842 1.00 0.01 ATOM 3152 N ASN A 347 24.938 −10.392 5.088 1.00 0.01 ATOM 3154 CA ASN A 347 26.273 −10.397 4.483 1.00 0.00 ATOM 3155 CB ASN A 347 26.212 −9.732 3.113 1.00 0.63 ATOM 3156 CG ASN A 347 25.547 −10.625 2.064 1.00 1.00 ATOM 3157 OD1 ASN A 347 24.760 −11.536 2.362 1.00 1.77 ATOM 3158 ND2 ASN A 347 25.894 −10.349 0.820 1.00 1.70 ATOM 3161 C ASN A 347 27.327 −9.696 5.322 1.00 0.01 ATOM 3162 O ASN A 347 28.491 −10.091 5.227 1.00 0.01 ATOM 3163 N ALA A 348 26.896 −8.925 6.309 1.00 0.01 ATOM 3165 CA ALA A 348 27.825 −8.229 7.205 1.00 0.01 ATOM 3166 CB ALA A 348 27.092 −7.043 7.822 1.00 0.19 ATOM 3167 C ALA A 348 28.345 −9.130 8.324 1.00 0.01 ATOM 3168 O ALA A 348 29.375 −8.827 8.939 1.00 0.00 ATOM 3169 N PHE A 349 27.681 −10.253 8.543 1.00 0.01 ATOM 3171 CA PHE A 349 28.155 −11.202 9.548 1.00 0.02 ATOM 3172 CB PHE A 349 26.947 −11.846 10.218 1.00 0.01 ATOM 3173 CG PHE A 349 25.980 −10.840 10.834 1.00 0.02 ATOM 3174 CD1 PHE A 349 26.446 −9.880 11.723 1.00 0.01 ATOM 3175 CE1 PHE A 349 25.569 −8.957 12.275 1.00 0.01 ATOM 3176 CZ PHE A 349 24.223 −8.998 11.944 1.00 0.00 ATOM 3177 CE2 PHE A 349 23.755 −9.966 11.065 1.00 0.01 ATOM 3178 CD2 PHE A 349 24.631 −10.888 10.511 1.00 0.00 ATOM 3179 C PHE A 349 28.996 −12.263 8.859 1.00 0.00 ATOM 3180 O PHE A 349 29.996 −12.742 9.402 1.00 0.02 ATOM 3181 N ASP A 350 28.658 −12.488 7.599 1.00 0.01 ATOM 3183 CA ASP A 350 29.393 −13.425 6.745 1.00 0.00 ATOM 3184 CB ASP A 350 28.670 −13.529 5.406 1.00 1.52 ATOM 3185 CG ASP A 350 27.237 −14.027 5.533 1.00 2.27 ATOM 3186 OD1 ASP A 350 26.987 −14.857 6.399 1.00 2.78 ATOM 3187 OD2 ASP A 350 26.428 −13.632 4.696 1.00 2.74 ATOM 3188 C ASP A 350 30.820 −12.944 6.481 1.00 0.01 ATOM 3189 O ASP A 350 31.122 −11.751 6.581 1.00 0.02 ATOM 3190 N LYS A 351 31.646 −13.860 5.996 1.00 0.02 ATOM 3192 CA LYS A 351 33.055 −13.558 5.674 1.00 0.01 ATOM 3193 CB LYS A 351 33.795 −14.881 5.543 1.00 0.65 ATOM 3194 CG LYS A 351 33.604 −15.760 6.772 1.00 1.13 ATOM 3195 CD LYS A 351 34.251 −17.123 6.564 1.00 1.37 ATOM 3196 CE LYS A 351 33.628 −17.836 5.368 1.00 2.60 ATOM 3197 NZ LYS A 351 34.266 −19.142 5.136 1.00 3.15 ATOM 3198 C LYS A 351 33.213 −12.806 4.350 1.00 0.02 ATOM 3199 O LYS A 351 34.297 −12.309 4.023 1.00 0.01 ATOM 3200 N ASN A 352 32.114 −12.692 3.627 1.00 0.02 ATOM 3202 CA ASN A 352 32.049 −11.964 2.361 1.00 0.00 ATOM 3203 CB ASN A 352 31.173 −12.759 1.390 1.00 0.51 ATOM 3204 CG ASN A 352 29.869 −13.231 2.040 1.00 1.28 ATOM 3205 OD1 ASN A 352 29.796 −14.333 2.595 1.00 1.84 ATOM 3206 ND2 ASN A 352 28.858 −12.381 1.990 1.00 2.06 ATOM 3209 C ASN A 352 31.522 −10.537 2.541 1.00 0.00 ATOM 3210 O ASN A 352 30.997 −9.951 1.581 1.00 0.01 ATOM 3211 N ALA A 353 31.494 −10.066 3.777 1.00 0.01 ATOM 3213 CA ALA A 353 31.074 −8.692 4.047 1.00 0.01 ATOM 3214 CB ALA A 353 31.097 −8.473 5.551 1.00 0.01 ATOM 3215 C ALA A 353 31.997 −7.683 3.379 1.00 0.01 ATOM 3216 O ALA A 353 33.229 −7.781 3.429 1.00 0.02 ATOM 3217 N ASP A 354 31.385 −6.708 2.728 1.00 0.01 ATOM 3219 CA ASP A 354 32.164 −5.651 2.088 1.00 0.01 ATOM 3220 CB ASP A 354 31.396 −5.129 0.870 1.00 0.01 ATOM 3221 CG ASP A 354 32.212 −4.138 0.030 1.00 0.00 ATOM 3222 OD1 ASP A 354 32.848 −3.267 0.615 1.00 0.01 ATOM 3223 OD2 ASP A 354 32.008 −4.141 −1.174 1.00 0.00 ATOM 3224 C ASP A 354 32.385 −4.542 3.103 1.00 0.00 ATOM 3225 O ASP A 354 31.565 −3.630 3.230 1.00 0.01 ATOM 3226 N PHE A 355 33.507 −4.604 3.793 1.00 0.02 ATOM 3228 CA PHE A 355 33.843 −3.552 4.751 1.00 0.02 ATOM 3229 CB PHE A 355 34.107 −4.179 6.113 1.00 1.78 ATOM 3230 CG PHE A 355 32.846 −4.653 6.834 1.00 2.43 ATOM 3231 CD1 PHE A 355 32.859 −5.839 7.555 1.00 3.08 ATOM 3232 CE1 PHE A 355 31.718 −6.265 8.221 1.00 3.90 ATOM 3233 CZ PHE A 355 30.559 −5.504 8.164 1.00 4.07 ATOM 3234 CE2 PHE A 355 30.542 −4.318 7.443 1.00 3.51 ATOM 3235 CD2 PHE A 355 31.686 −3.893 6.779 1.00 2.73 ATOM 3236 C PHE A 355 35.046 −2.749 4.275 1.00 0.02 ATOM 3237 O PHE A 355 35.975 −2.488 5.052 1.00 0.02 ATOM 3238 N SER A 356 34.885 −2.144 3.107 1.00 0.02 ATOM 3240 CA SER A 356 35.978 −1.419 2.434 1.00 0.00 ATOM 3241 CB SER A 356 35.684 −1.364 0.940 1.00 0.01 ATOM 3242 OG SER A 356 34.507 −0.591 0.750 1.00 0.01 ATOM 3243 C SER A 356 36.196 0.009 2.949 1.00 0.02 ATOM 3244 O SER A 356 37.063 0.728 2.438 1.00 0.01 ATOM 3245 N GLY A 357 35.391 0.425 3.912 1.00 0.00 ATOM 3247 CA GLY A 357 35.604 1.702 4.590 1.00 0.01 ATOM 3248 C GLY A 357 36.279 1.465 5.937 1.00 0.02 ATOM 3249 O GLY A 357 36.934 2.363 6.484 1.00 0.02 ATOM 3250 N ILE A 358 36.160 0.241 6.429 1.00 0.00 ATOM 3252 CA ILE A 358 36.753 −0.120 7.716 1.00 0.01 ATOM 3253 CB ILE A 358 35.915 −1.222 8.354 1.00 1.15 ATOM 3254 CG2 ILE A 358 36.415 −1.521 9.764 1.00 1.49 ATOM 3255 CG1 ILE A 358 34.440 −0.854 8.382 1.00 1.60 ATOM 3256 CD1 ILE A 358 33.617 −1.909 9.109 1.00 2.08 ATOM 3257 C ILE A 358 38.158 −0.666 7.519 1.00 0.02 ATOM 3258 O ILE A 358 39.102 −0.216 8.178 1.00 0.02 ATOM 3259 N ALA A 359 38.280 −1.586 6.576 1.00 0.02 ATOM 3261 CA ALA A 359 39.550 −2.261 6.296 1.00 0.00 ATOM 3262 CB ALA A 359 39.821 −3.283 7.395 1.00 0.83 ATOM 3263 C ALA A 359 39.467 −2.976 4.953 1.00 0.02 ATOM 3264 O ALA A 359 39.356 −4.210 4.917 1.00 0.01 ATOM 3265 N LYS A 360 39.799 −2.242 3.903 1.00 0.00 ATOM 3267 CA LYS A 360 39.608 −2.681 2.507 1.00 0.00 ATOM 3268 CB LYS A 360 39.945 −1.487 1.618 1.00 1.13 ATOM 3269 CG LYS A 360 41.351 −0.963 1.906 1.00 1.56 ATOM 3270 CD LYS A 360 41.759 0.129 0.926 1.00 2.17 ATOM 3271 CE LYS A 360 40.773 1.289 0.948 1.00 3.04 ATOM 3272 NZ LYS A 360 41.170 2.337 −0.004 1.00 3.66 ATOM 3273 C LYS A 360 40.437 −3.877 2.012 1.00 0.02 ATOM 3274 O LYS A 360 40.140 −4.394 0.930 1.00 0.02 ATOM 3275 N ARG A 361 41.421 −4.340 2.765 1.00 0.02 ATOM 3277 CA ARG A 361 42.187 −5.507 2.324 1.00 0.00 ATOM 3278 CB ARG A 361 43.662 −5.131 2.299 1.00 1.38 ATOM 3279 CG ARG A 361 44.165 −4.694 3.668 1.00 1.86 ATOM 3280 CD ARG A 361 45.493 −3.965 3.534 1.00 2.43 ATOM 3281 NE ARG A 361 45.300 −2.722 2.770 1.00 3.31 ATOM 3282 CZ ARG A 361 46.256 −1.808 2.594 1.00 4.21 ATOM 3283 NH1 ARG A 361 45.992 −0.690 1.914 1.00 5.21 ATOM 3284 NH2 ARG A 361 47.469 −2.001 3.118 1.00 4.31 ATOM 3285 C ARG A 361 41.939 −6.728 3.211 1.00 0.02 ATOM 3286 O ARG A 361 42.483 −7.809 2.956 1.00 0.00 ATOM 3287 N ASP A 362 41.107 −6.562 4.227 1.00 0.02 ATOM 3289 CA ASP A 362 40.847 −7.655 5.171 1.00 0.01 ATOM 3290 CB ASP A 362 41.005 −7.113 6.592 1.00 0.80 ATOM 3291 CG ASP A 362 42.408 −6.547 6.824 1.00 0.66 ATOM 3292 OD1 ASP A 362 43.357 −7.142 6.338 1.00 0.78 ATOM 3293 OD2 ASP A 362 42.506 −5.501 7.457 1.00 1.43 ATOM 3294 C ASP A 362 39.432 −8.207 4.989 1.00 0.02 ATOM 3295 O ASP A 362 38.485 −7.445 4.769 1.00 0.02 ATOM 3296 N SER A 363 39.293 −9.522 5.072 1.00 0.01 ATOM 3298 CA SER A 363 37.955 −10.144 5.029 1.00 0.02 ATOM 3299 CB SER A 363 38.077 −11.595 4.580 1.00 0.34 ATOM 3300 OG SER A 363 38.580 −11.595 3.250 1.00 0.54 ATOM 3301 C SER A 363 37.296 −10.070 6.407 1.00 0.02 ATOM 3302 O SER A 363 37.486 −10.940 7.268 1.00 0.00 ATOM 3303 N LEU A 364 36.566 −8.987 6.610 1.00 0.01 ATOM 3305 CA LEU A 364 36.018 −8.664 7.926 1.00 0.01 ATOM 3306 CB LEU A 364 36.083 −7.147 8.066 1.00 0.00 ATOM 3307 CG LEU A 364 35.857 −6.672 9.495 1.00 0.02 ATOM 3308 CD1 LEU A 364 36.971 −7.164 10.406 1.00 0.01 ATOM 3309 CD2 LEU A 364 35.790 −5.153 9.540 1.00 0.01 ATOM 3310 C LEU A 364 34.586 −9.173 8.085 1.00 0.00 ATOM 3311 O LEU A 364 33.715 −8.941 7.243 1.00 0.00 ATOM 3312 N GLN A 365 34.374 −9.874 9.186 1.00 0.01 ATOM 3314 CA GLN A 365 33.063 −10.445 9.512 1.00 0.02 ATOM 3315 CB GLN A 365 33.195 −11.960 9.376 1.00 0.93 ATOM 3316 CG GLN A 365 34.513 −12.447 9.973 1.00 0.96 ATOM 3317 CD GLN A 365 34.684 −13.954 9.795 1.00 1.26 ATOM 3318 OE1 GLN A 365 33.725 −14.728 9.900 1.00 1.74 ATOM 3319 NE2 GLN A 365 35.906 −14.343 9.476 1.00 1.77 ATOM 3322 C GLN A 365 32.612 −10.062 10.924 1.00 0.01 ATOM 3323 O GLN A 365 33.414 −10.108 11.873 1.00 0.02 ATOM 3324 N VAL A 366 31.372 −9.605 11.042 1.00 0.02 ATOM 3326 CA VAL A 366 30.808 −9.330 12.373 1.00 0.02 ATOM 3327 CB VAL A 366 29.611 −8.391 12.278 1.00 0.05 ATOM 3328 CG1 VAL A 366 29.051 −8.088 13.665 1.00 0.07 ATOM 3329 CG2 VAL A 366 29.998 −7.092 11.582 1.00 0.09 ATOM 3330 C VAL A 366 30.427 −10.653 13.030 1.00 0.01 ATOM 3331 O VAL A 366 29.388 −11.264 12.747 1.00 0.01 ATOM 3332 N SER A 367 31.218 −10.967 14.038 1.00 0.01 ATOM 3334 CA SER A 367 31.284 −12.311 14.608 1.00 0.01 ATOM 3335 CB SER A 367 32.695 −12.811 14.376 1.00 0.02 ATOM 3336 OG SER A 367 33.550 −11.755 14.784 1.00 0.02 ATOM 3337 C SER A 367 30.928 −12.353 16.091 1.00 0.00 ATOM 3338 O SER A 367 31.261 −13.332 16.778 1.00 0.01 ATOM 3339 N LYS A 368 30.497 −11.208 16.600 1.00 0.01 ATOM 3341 CA LYS A 368 29.771 −11.178 17.877 1.00 0.01 ATOM 3342 CB LYS A 368 30.719 −11.360 19.055 1.00 0.01 ATOM 3343 CG LYS A 368 29.919 −11.731 20.299 1.00 0.01 ATOM 3344 CD LYS A 368 29.108 −12.999 20.048 1.00 0.01 ATOM 3345 CE LYS A 368 28.171 −13.322 21.206 1.00 0.01 ATOM 3346 NZ LYS A 368 28.916 −13.569 22.447 1.00 0.02 ATOM 3347 C LYS A 368 28.985 −9.875 18.041 1.00 0.02 ATOM 3348 O LYS A 368 29.441 −8.950 18.725 1.00 0.01 ATOM 3349 N ALA A 369 27.850 −9.782 17.369 1.00 0.01 ATOM 3351 CA ALA A 369 26.963 −8.618 17.546 1.00 0.01 ATOM 3352 CB ALA A 369 26.238 −8.345 16.235 1.00 0.01 ATOM 3353 C ALA A 369 25.949 −8.864 18.664 1.00 0.01 ATOM 3354 O ALA A 369 25.158 −9.812 18.592 1.00 0.02 ATOM 3355 N THR A 370 25.939 −7.993 19.662 1.00 0.01 ATOM 3357 CA THR A 370 25.101 −8.219 20.852 1.00 0.01 ATOM 3358 CB THR A 370 25.947 −8.954 21.890 1.00 0.48 ATOM 3359 OG1 THR A 370 26.430 −10.157 21.304 1.00 1.35 ATOM 3360 CG2 THR A 370 25.143 −9.328 23.131 1.00 1.19 ATOM 3361 C THR A 370 24.538 −6.927 21.465 1.00 0.01 ATOM 3362 O THR A 370 25.275 −6.052 21.941 1.00 0.01 ATOM 3363 N HIS A 371 23.217 −6.866 21.486 1.00 0.01 ATOM 3365 CA HIS A 371 22.455 −5.767 22.092 1.00 0.01 ATOM 3366 CB HIS A 371 21.330 −5.439 21.104 1.00 0.01 ATOM 3367 CG HIS A 371 20.200 −4.573 21.621 1.00 0.01 ATOM 3368 ND1 HIS A 371 20.198 −3.234 21.750 1.00 0.01 ATOM 3370 CE1 HIS A 371 19.017 −2.838 22.261 1.00 0.01 ATOM 3371 NE2 HIS A 371 18.262 −3.943 22.450 1.00 0.01 ATOM 3372 CD2 HIS A 371 18.976 −5.020 22.056 1.00 0.01 ATOM 3373 C HIS A 371 21.887 −6.179 23.457 1.00 0.01 ATOM 3374 O HIS A 371 21.726 −7.377 23.718 1.00 0.01 ATOM 3375 N LYS A 372 21.692 −5.220 24.351 1.00 0.01 ATOM 3377 CA LYS A 372 20.961 −5.502 25.600 1.00 0.01 ATOM 3378 CB LYS A 372 21.890 −6.097 26.651 1.00 0.01 ATOM 3379 CG LYS A 372 21.111 −6.441 27.917 1.00 0.01 ATOM 3380 CD LYS A 372 22.006 −6.977 29.023 1.00 0.01 ATOM 3381 CE LYS A 372 21.187 −7.286 30.270 1.00 0.01 ATOM 3382 NZ LYS A 372 22.038 −7.707 31.393 1.00 0.00 ATOM 3383 C LYS A 372 20.295 −4.252 26.174 1.00 0.01 ATOM 3384 O LYS A 372 20.979 −3.322 26.625 1.00 0.01 ATOM 3385 N ALA A 373 18.972 −4.272 26.212 1.00 0.02 ATOM 3387 CA ALA A 373 18.209 −3.152 26.778 1.00 0.01 ATOM 3388 CB ALA A 373 17.019 −2.865 25.872 1.00 0.01 ATOM 3389 C ALA A 373 17.735 −3.444 28.206 1.00 0.01 ATOM 3390 O ALA A 373 17.267 −4.549 28.512 1.00 0.01 ATOM 3391 N VAL A 374 17.925 −2.470 29.083 1.00 0.01 ATOM 3393 CA VAL A 374 17.524 −2.612 30.496 1.00 0.01 ATOM 3394 CB VAL A 374 18.783 −2.646 31.364 1.00 0.01 ATOM 3395 CG1 VAL A 374 18.454 −3.123 32.775 1.00 0.01 ATOM 3396 CG2 VAL A 374 19.876 −3.526 30.769 1.00 0.01 ATOM 3397 C VAL A 374 16.649 −1.435 30.951 1.00 0.01 ATOM 3398 O VAL A 374 17.059 −0.273 30.841 1.00 0.01 ATOM 3399 N LEU A 375 15.489 −1.738 31.512 1.00 0.02 ATOM 3401 CA LEU A 375 14.565 −0.695 31.983 1.00 0.01 ATOM 3402 CB LEU A 375 13.288 −0.778 31.165 1.00 0.01 ATOM 3403 CG LEU A 375 12.297 0.324 31.521 1.00 0.01 ATOM 3404 CD1 LEU A 375 12.882 1.700 31.229 1.00 0.01 ATOM 3405 CD2 LEU A 375 10.991 0.130 30.767 1.00 0.00 ATOM 3406 C LEU A 375 14.170 −0.871 33.443 1.00 0.01 ATOM 3407 O LEU A 375 13.437 −1.802 33.796 1.00 0.02 ATOM 3408 N ASP A 376 14.540 0.096 34.262 1.00 0.02 ATOM 3410 CA ASP A 376 14.090 0.092 35.653 1.00 0.02 ATOM 3411 CB ASP A 376 15.157 0.722 36.539 1.00 0.48 ATOM 3412 CG ASP A 376 14.646 0.786 37.978 1.00 1.01 ATOM 3413 OD1 ASP A 376 14.721 −0.232 38.650 1.00 1.53 ATOM 3414 OD2 ASP A 376 14.109 1.826 38.344 1.00 1.55 ATOM 3415 C ASP A 376 12.779 0.859 35.806 1.00 0.01 ATOM 3416 O ASP A 376 12.756 2.099 35.767 1.00 0.02 ATOM 3417 N VAL A 377 11.702 0.116 36.010 1.00 0.01 ATOM 3419 CA VAL A 377 10.405 0.746 36.276 1.00 0.01 ATOM 3420 CB VAL A 377 9.297 −0.111 35.676 1.00 0.54 ATOM 3421 CG1 VAL A 377 7.930 0.537 35.872 1.00 0.52 ATOM 3422 CG2 VAL A 377 9.551 −0.371 34.197 1.00 0.68 ATOM 3423 C VAL A 377 10.184 0.903 37.779 1.00 0.02 ATOM 3424 O VAL A 377 9.720 −0.020 38.459 1.00 0.00 ATOM 3425 N SER A 378 10.590 2.055 38.282 1.00 0.01 ATOM 3427 CA SER A 378 10.411 2.405 39.699 1.00 0.02 ATOM 3428 CB SER A 378 11.661 3.126 40.188 1.00 0.91 ATOM 3429 OG SER A 378 11.816 4.305 39.406 1.00 1.28 ATOM 3430 C SER A 378 9.200 3.317 39.861 1.00 0.00 ATOM 3431 O SER A 378 8.473 3.549 38.890 1.00 0.01 ATOM 3432 N GLU A 379 8.933 3.762 41.081 1.00 0.02 ATOM 3434 CA GLU A 379 7.850 4.742 41.287 1.00 0.02 ATOM 3435 CB GLU A 379 7.299 4.673 42.710 1.00 0.02 ATOM 3436 CG GLU A 379 6.699 3.326 43.083 1.00 0.02 ATOM 3437 CD GLU A 379 7.621 2.653 44.083 1.00 0.02 ATOM 3438 OE1 GLU A 379 8.796 3.005 44.070 1.00 0.02 ATOM 3439 OE2 GLU A 379 7.165 1.780 44.802 1.00 0.02 ATOM 3440 C GLU A 379 8.405 6.144 41.093 1.00 0.00 ATOM 3441 O GLU A 379 7.702 7.072 40.671 1.00 0.02 ATOM 3442 N GLU A 380 9.660 6.274 41.489 1.00 0.01 ATOM 3444 CA GLU A 380 10.451 7.489 41.291 1.00 0.00 ATOM 3445 CB GLU A 380 10.535 8.262 42.607 1.00 1.77 ATOM 3446 CG GLU A 380 9.195 8.834 43.055 1.00 2.64 ATOM 3447 CD GLU A 380 9.342 9.500 44.420 1.00 3.00 ATOM 3448 OE1 GLU A 380 9.953 8.886 45.286 1.00 3.53 ATOM 3449 OE2 GLU A 380 8.737 10.548 44.608 1.00 3.27 ATOM 3450 C GLU A 380 11.858 7.081 40.876 1.00 0.02 ATOM 3451 O GLU A 380 12.452 6.198 41.508 1.00 0.02 ATOM 3452 N GLY A 381 12.322 7.613 39.759 1.00 0.02 ATOM 3454 CA GLY A 381 13.704 7.367 39.336 1.00 0.02 ATOM 3455 C GLY A 381 14.645 8.022 40.337 1.00 0.01 ATOM 3456 O GLY A 381 15.354 7.349 41.094 1.00 0.02 ATOM 3457 N THR A 382 14.639 9.342 40.330 1.00 0.01 ATOM 3459 CA THR A 382 15.374 10.092 41.346 1.00 0.01 ATOM 3460 CB THR A 382 15.537 11.538 40.880 1.00 0.02 ATOM 3461 OG1 THR A 382 16.095 12.288 41.953 1.00 0.01 ATOM 3462 CG2 THR A 382 14.199 12.173 40.517 1.00 0.02 ATOM 3463 C THR A 382 14.621 10.051 42.671 1.00 0.00 ATOM 3464 O THR A 382 13.390 9.952 42.703 1.00 0.02 ATOM 3465 N GLU A 383 15.377 10.089 43.756 1.00 0.01 ATOM 3467 CA GLU A 383 14.787 10.186 45.091 1.00 0.01 ATOM 3468 CB GLU A 383 15.725 9.519 46.089 1.00 0.02 ATOM 3469 CG GLU A 383 15.867 8.028 45.806 1.00 0.00 ATOM 3470 CD GLU A 383 16.883 7.415 46.763 1.00 0.01 ATOM 3471 OE1 GLU A 383 18.064 7.506 46.455 1.00 0.02 ATOM 3472 OE2 GLU A 383 16.468 6.881 47.781 1.00 0.01 ATOM 3473 C GLU A 383 14.521 11.633 45.523 1.00 0.01 ATOM 3474 O GLU A 383 14.041 11.842 46.642 1.00 0.01 ATOM 3475 N ALA A 384 14.842 12.614 44.689 1.00 0.01 ATOM 3477 CA ALA A 384 14.536 14.000 45.044 1.00 0.01 ATOM 3478 CB ALA A 384 15.301 14.930 44.110 1.00 0.01 ATOM 3479 C ALA A 384 13.039 14.246 44.909 1.00 0.01 ATOM 3480 O ALA A 384 12.393 13.719 43.996 1.00 0.02 ATOM 3481 N THR A 385 12.506 15.026 45.832 1.00 0.01 ATOM 3483 CA THR A 385 11.069 15.321 45.844 1.00 0.01 ATOM 3484 CB THR A 385 10.739 16.039 47.151 1.00 0.38 ATOM 3485 OG1 THR A 385 11.064 15.161 48.221 1.00 1.48 ATOM 3486 CG2 THR A 385 9.258 16.388 47.268 1.00 1.35 ATOM 3487 C THR A 385 10.675 16.184 44.648 1.00 0.02 ATOM 3488 O THR A 385 11.220 17.273 44.436 1.00 0.01 ATOM 3489 N ALA A 386 9.765 15.650 43.850 1.00 0.00 ATOM 3491 CA ALA A 386 9.233 16.371 42.691 1.00 0.02 ATOM 3492 CB ALA A 386 8.398 15.407 41.855 1.00 0.42 ATOM 3493 C ALA A 386 8.369 17.551 43.126 1.00 0.03 ATOM 3494 O ALA A 386 7.949 17.640 44.287 1.00 0.02 ATOM 3495 N ALA A 387 8.167 18.475 42.201 1.00 0.01 ATOM 3497 CA ALA A 387 7.315 19.643 42.456 1.00 0.01 ATOM 3498 CB ALA A 387 7.267 20.502 41.198 1.00 0.02 ATOM 3499 C ALA A 387 5.903 19.213 42.841 1.00 0.01 ATOM 3500 O ALA A 387 5.370 18.228 42.314 1.00 0.02 ATOM 3501 N THR A 388 5.314 19.953 43.764 1.00 0.02 ATOM 3503 CA THR A 388 3.986 19.603 44.282 1.00 0.01 ATOM 3504 CB THR A 388 3.830 20.215 45.670 1.00 0.52 ATOM 3505 OG1 THR A 388 4.892 19.727 46.479 1.00 0.64 ATOM 3506 CG2 THR A 388 2.511 19.810 46.324 1.00 0.69 ATOM 3507 C THR A 388 2.866 20.085 43.362 1.00 0.02 ATOM 3508 O THR A 388 2.391 21.223 43.457 1.00 0.01 ATOM 3509 N THR A 389 2.504 19.221 42.431 1.00 0.01 ATOM 3511 CA THR A 389 1.383 19.495 41.527 1.00 0.02 ATOM 3512 CB THR A 389 1.577 18.714 40.233 1.00 0.16 ATOM 3513 OG1 THR A 389 1.515 17.323 40.528 1.00 0.18 ATOM 3514 CG2 THR A 389 2.927 19.015 39.592 1.00 0.12 ATOM 3515 C THR A 389 0.072 19.085 42.186 1.00 0.02 ATOM 3516 O THR A 389 0.004 18.073 42.891 1.00 0.02 ATOM 3517 N THR A 390 −0.958 19.883 41.971 1.00 0.01 ATOM 3519 CA THR A 390 −2.260 19.577 42.572 1.00 0.02 ATOM 3520 CB THR A 390 −2.500 20.548 43.722 1.00 0.38 ATOM 3521 OG1 THR A 390 −1.396 20.444 44.612 1.00 1.27 ATOM 3522 CG2 THR A 390 −3.772 20.210 44.494 1.00 1.29 ATOM 3523 C THR A 390 −3.383 19.680 41.545 1.00 0.01 ATOM 3524 O THR A 390 −3.969 20.749 41.338 1.00 0.02 ATOM 3525 N LYS A 391 −3.640 18.572 40.874 1.00 0.02 ATOM 3527 CA LYS A 391 −4.725 18.537 39.890 1.00 0.01 ATOM 3528 CB LYS A 391 −4.297 17.698 38.695 1.00 0.84 ATOM 3529 CG LYS A 391 −3.093 18.351 38.027 1.00 1.32 ATOM 3530 CD LYS A 391 −2.706 17.653 36.732 1.00 2.13 ATOM 3531 CE LYS A 391 −1.480 18.312 36.110 1.00 2.60 ATOM 3532 NZ LYS A 391 −0.340 18.271 37.038 1.00 3.28 ATOM 3533 C LYS A 391 −6.011 18.011 40.516 1.00 0.02 ATOM 3534 O LYS A 391 −6.000 17.074 41.322 1.00 0.01 ATOM 3535 N PHE A 392 −7.107 18.653 40.155 1.00 0.01 ATOM 3537 CA PHE A 392 −8.408 18.332 40.749 1.00 0.01 ATOM 3538 CB PHE A 392 −9.331 19.528 40.552 1.00 0.12 ATOM 3539 CG PHE A 392 −8.787 20.807 41.183 1.00 0.14 ATOM 3540 CD1 PHE A 392 −8.513 21.914 40.390 1.00 0.22 ATOM 3541 CE1 PHE A 392 −8.010 23.074 40.966 1.00 0.31 ATOM 3542 CZ PHE A 392 −7.782 23.126 42.336 1.00 0.36 ATOM 3543 CE2 PHE A 392 −8.058 22.020 43.128 1.00 0.33 ATOM 3544 CD2 PHE A 392 −8.560 20.861 42.552 1.00 0.20 ATOM 3545 C PHE A 392 −9.025 17.075 40.146 1.00 0.02 ATOM 3546 O PHE A 392 −9.271 16.983 38.937 1.00 0.02 ATOM 3547 N ILE A 393 −9.234 16.102 41.016 1.00 0.02 ATOM 3549 CA ILE A 393 −9.840 14.829 40.625 1.00 0.02 ATOM 3550 CB ILE A 393 −9.109 13.720 41.379 1.00 0.02 ATOM 3551 CG2 ILE A 393 −9.669 12.341 41.044 1.00 0.00 ATOM 3552 CG1 ILE A 393 −7.618 13.771 41.066 1.00 0.02 ATOM 3553 CD1 ILE A 393 −6.851 12.724 41.862 1.00 0.00 ATOM 3554 C ILE A 393 −11.330 14.807 40.954 1.00 0.02 ATOM 3555 O ILE A 393 −11.736 15.082 42.089 1.00 0.02 ATOM 3556 N VAL A 394 −12.137 14.553 39.937 1.00 0.02 ATOM 3558 CA VAL A 394 −13.579 14.400 40.147 1.00 0.02 ATOM 3559 CB VAL A 394 −14.274 14.489 38.787 1.00 1.05 ATOM 3560 CG1 VAL A 394 −15.768 14.192 38.854 1.00 1.37 ATOM 3561 CG2 VAL A 394 −14.052 15.872 38.188 1.00 1.35 ATOM 3562 C VAL A 394 −13.848 13.075 40.863 1.00 0.02 ATOM 3563 O VAL A 394 −13.339 12.021 40.472 1.00 0.00 ATOM 3564 N ARG A 395 −14.678 13.150 41.893 1.00 0.02 ATOM 3566 CA ARG A 395 −14.955 12.017 42.792 1.00 0.02 ATOM 3567 CB ARG A 395 −15.414 12.644 44.108 1.00 1.17 ATOM 3568 CG ARG A 395 −15.493 11.662 45.273 1.00 1.69 ATOM 3569 CD ARG A 395 −16.026 12.350 46.524 1.00 2.28 ATOM 3570 NE ARG A 395 −17.365 12.912 46.275 1.00 2.83 ATOM 3571 CZ ARG A 395 −17.679 14.191 46.492 1.00 3.34 ATOM 3572 NH1 ARG A 395 −16.753 15.040 46.945 1.00 3.83 ATOM 3573 NH2 ARG A 395 −18.915 14.626 46.237 1.00 3.68 ATOM 3574 C ARG A 395 −16.021 11.035 42.266 1.00 0.02 ATOM 3575 O ARG A 395 −16.340 10.047 42.939 1.00 0.02 ATOM 3576 N SER A 396 −16.547 11.286 41.077 1.00 0.01 ATOM 3578 CA SER A 396 −17.573 10.408 40.497 1.00 0.01 ATOM 3579 CB SER A 396 −18.008 10.956 39.140 1.00 0.02 ATOM 3580 OG SER A 396 −16.889 10.942 38.261 1.00 0.00 ATOM 3581 C SER A 396 −17.044 8.988 40.324 1.00 0.02 ATOM 3582 O SER A 396 −15.959 8.777 39.774 1.00 0.01 ATOM 3583 N LYS A 397 −17.903 8.025 40.616 1.00 0.01 ATOM 3585 CA LYS A 397 −17.504 6.608 40.597 1.00 0.01 ATOM 3586 CB LYS A 397 −18.483 5.845 41.476 1.00 0.53 ATOM 3587 CG LYS A 397 −18.443 6.404 42.894 1.00 0.99 ATOM 3588 CD LYS A 397 −19.445 5.717 43.811 1.00 1.27 ATOM 3589 CE LYS A 397 −19.381 6.301 45.218 1.00 1.17 ATOM 3590 NZ LYS A 397 −19.656 7.748 45.199 1.00 1.92 ATOM 3591 C LYS A 397 −17.456 5.995 39.195 1.00 0.02 ATOM 3592 O LYS A 397 −17.020 4.854 39.020 1.00 0.02 ATOM 3593 N ASP A 398 −17.852 6.773 38.200 1.00 0.53 ATOM 3595 CA ASP A 398 −17.694 6.369 36.803 1.00 1.04 ATOM 3596 CB ASP A 398 −18.868 6.954 36.019 1.00 1.51 ATOM 3597 CG ASP A 398 −18.826 6.533 34.554 1.00 1.90 ATOM 3598 OD1 ASP A 398 −18.447 7.362 33.738 1.00 2.32 ATOM 3599 OD2 ASP A 398 −19.301 5.443 34.265 1.00 2.30 ATOM 3600 C ASP A 398 −16.367 6.900 36.246 1.00 1.10 ATOM 3601 O ASP A 398 −15.881 6.413 35.218 1.00 1.33 ATOM 3602 N GLY A 399 −15.729 7.791 36.992 1.00 0.35 ATOM 3604 CA GLY A 399 −14.472 8.400 36.545 1.00 0.43 ATOM 3605 C GLY A 399 −13.299 7.483 36.865 1.00 0.38 ATOM 3606 O GLY A 399 −12.968 6.607 36.055 1.00 0.43 ATOM 3607 N PRO A 400 −12.657 7.729 38.000 1.00 0.02 ATOM 3608 CA PRO A 400 −11.593 6.852 38.509 1.00 0.00 ATOM 3609 CB PRO A 400 −11.054 7.548 39.717 1.00 0.02 ATOM 3610 CG PRO A 400 −11.911 8.768 40.023 1.00 0.03 ATOM 3611 CD PRO A 400 −12.932 8.851 38.902 1.00 0.03 ATOM 3612 C PRO A 400 −12.127 5.475 38.879 1.00 0.01 ATOM 3613 O PRO A 400 −12.521 5.237 40.026 1.00 0.00 ATOM 3614 N SER A 401 −12.080 4.568 37.919 1.00 0.02 ATOM 3616 CA SER A 401 −12.717 3.268 38.096 1.00 0.01 ATOM 3617 CB SER A 401 −13.842 3.213 37.071 1.00 2.33 ATOM 3618 OG SER A 401 −14.658 4.361 37.292 1.00 3.03 ATOM 3619 C SER A 401 −11.770 2.090 37.914 1.00 0.02 ATOM 3620 O SER A 401 −10.675 2.214 37.356 1.00 0.01 ATOM 3621 N TYR A 402 −12.326 0.933 38.231 1.00 1.42 ATOM 3623 CA TYR A 402 −11.639 −0.367 38.268 1.00 1.57 ATOM 3624 CB TYR A 402 −12.601 −1.427 38.863 1.00 1.62 ATOM 3625 CG TYR A 402 −14.106 −1.543 38.474 1.00 2.37 ATOM 3626 CD1 TYR A 402 −14.922 −2.337 39.277 1.00 3.23 ATOM 3627 CE1 TYR A 402 −16.272 −2.486 38.985 1.00 4.33 ATOM 3628 CZ TYR A 402 −16.815 −1.847 37.882 1.00 4.54 ATOM 3629 OH TYR A 402 −18.149 −2.013 37.577 1.00 5.70 ATOM 3630 CE2 TYR A 402 −16.013 −1.065 37.065 1.00 3.73 ATOM 3631 CD2 TYR A 402 −14.663 −0.922 37.358 1.00 2.65 ATOM 3632 C TYR A 402 −10.998 −0.872 36.963 1.00 1.61 ATOM 3633 O TYR A 402 −9.774 −0.749 36.800 1.00 2.21 ATOM 3634 N PHE A 403 −11.818 −1.232 35.982 1.00 1.32 ATOM 3636 CA PHE A 403 −11.365 −2.043 34.842 1.00 1.54 ATOM 3637 CB PHE A 403 −12.592 −2.504 34.059 1.00 1.10 ATOM 3638 CG PHE A 403 −13.556 −3.460 34.757 1.00 1.20 ATOM 3639 CD1 PHE A 403 −14.908 −3.425 34.434 1.00 1.54 ATOM 3640 CE1 PHE A 403 −15.794 −4.301 35.048 1.00 2.04 ATOM 3641 CZ PHE A 403 −15.324 −5.218 35.978 1.00 2.06 ATOM 3642 CE2 PHE A 403 −13.972 −5.270 36.291 1.00 1.68 ATOM 3643 CD2 PHE A 403 −13.086 −4.394 35.674 1.00 1.35 ATOM 3644 C PHE A 403 −10.458 −1.342 33.840 1.00 1.40 ATOM 3645 O PHE A 403 −9.673 −2.022 33.171 1.00 2.11 ATOM 3646 N THR A 404 −10.380 −0.022 33.889 1.00 1.47 ATOM 3648 CA THR A 404 −9.645 0.688 32.838 1.00 1.83 ATOM 3649 CB THR A 404 −10.219 2.093 32.686 1.00 1.26 ATOM 3650 OG1 THR A 404 −9.975 2.812 33.891 1.00 1.46 ATOM 3651 CG2 THR A 404 −11.724 2.058 32.432 1.00 2.05 ATOM 3652 C THR A 404 −8.152 0.792 33.122 1.00 1.32 ATOM 3653 O THR A 404 −7.371 1.044 32.200 1.00 1.45 ATOM 3654 N VAL A 405 −7.747 0.528 34.352 1.00 0.00 ATOM 3656 CA VAL A 405 −6.363 0.806 34.721 1.00 0.02 ATOM 3657 CB VAL A 405 −6.383 2.110 35.488 1.00 0.02 ATOM 3658 CG1 VAL A 405 −6.272 3.287 34.531 1.00 0.02 ATOM 3659 CG2 VAL A 405 −7.660 2.182 36.311 1.00 0.02 ATOM 3660 C VAL A 405 −5.687 −0.287 35.537 1.00 0.01 ATOM 3661 O VAL A 405 −4.657 −0.029 36.177 1.00 0.00 ATOM 3662 N SER A 406 −6.268 −1.473 35.546 1.00 0.02 ATOM 3664 CA SER A 406 −5.611 −2.602 36.206 1.00 0.02 ATOM 3665 CB SER A 406 −6.655 −3.585 36.743 1.00 2.03 ATOM 3666 OG SER A 406 −7.513 −3.997 35.685 1.00 2.68 ATOM 3667 C SER A 406 −4.638 −3.287 35.242 1.00 0.02 ATOM 3668 O SER A 406 −5.017 −4.174 34.471 1.00 0.01 ATOM 3669 N PHE A 407 −3.364 −2.958 35.384 1.00 0.02 ATOM 3671 CA PHE A 407 −2.322 −3.488 34.494 1.00 0.02 ATOM 3672 CB PHE A 407 −1.268 −2.413 34.235 1.00 0.01 ATOM 3673 CG PHE A 407 −1.739 −1.291 33.311 1.00 0.02 ATOM 3674 CD1 PHE A 407 −2.349 −0.155 33.828 1.00 0.02 ATOM 3675 CE1 PHE A 407 −2.782 0.851 32.974 1.00 0.02 ATOM 3676 CZ PHE A 407 −2.598 0.725 31.603 1.00 0.02 ATOM 3677 CE2 PHE A 407 −1.977 −0.405 31.087 1.00 0.00 ATOM 3678 CD2 PHE A 407 −1.548 −1.412 31.941 1.00 0.01 ATOM 3679 C PHE A 407 −1.682 −4.760 35.045 1.00 0.02 ATOM 3680 O PHE A 407 −0.462 −4.949 34.979 1.00 0.02 ATOM 3681 N ASN A 408 −2.517 −5.611 35.619 1.00 0.02 ATOM 3683 CA ASN A 408 −2.054 −6.901 36.120 1.00 0.02 ATOM 3684 CB ASN A 408 −2.829 −7.317 37.375 1.00 0.02 ATOM 3685 CG ASN A 408 −4.294 −7.673 37.106 1.00 0.01 ATOM 3686 OD1 ASN A 408 −5.030 −6.943 36.432 1.00 0.01 ATOM 3687 ND2 ASN A 408 −4.709 −8.787 37.681 1.00 0.02 ATOM 3690 C ASN A 408 −2.170 −7.944 35.014 1.00 0.02 ATOM 3691 O ASN A 408 −1.639 −9.047 35.153 1.00 0.00 ATOM 3692 N ARG A 409 −2.855 −7.619 33.932 1.00 0.00 ATOM 3694 CA ARG A 409 −2.766 −8.471 32.743 1.00 0.01 ATOM 3695 CB ARG A 409 −4.057 −8.395 31.946 1.00 1.08 ATOM 3696 CG ARG A 409 −5.240 −8.876 32.774 1.00 1.16 ATOM 3697 CD ARG A 409 −6.500 −8.955 31.924 1.00 1.61 ATOM 3698 NE ARG A 409 −6.792 −7.655 31.304 1.00 1.42 ATOM 3699 CZ ARG A 409 −8.026 −7.155 31.217 1.00 1.93 ATOM 3700 NH1 ARG A 409 −9.060 −7.845 31.703 1.00 2.76 ATOM 3701 NH2 ARG A 409 −8.225 −5.967 30.641 1.00 2.23 ATOM 3702 C ARG A 409 −1.585 −8.014 31.893 1.00 0.00 ATOM 3703 O ARG A 409 −1.125 −6.876 32.038 1.00 0.01 ATOM 3704 N THR A 410 −1.078 −8.921 31.074 1.00 0.01 ATOM 3706 CA THR A 410 0.062 −8.630 30.187 1.00 0.01 ATOM 3707 CB THR A 410 0.301 −9.867 29.326 1.00 0.83 ATOM 3708 OG1 THR A 410 0.808 −10.886 30.181 1.00 1.17 ATOM 3709 CG2 THR A 410 1.328 −9.629 28.222 1.00 1.37 ATOM 3710 C THR A 410 −0.176 −7.399 29.309 1.00 0.01 ATOM 3711 O THR A 410 −1.183 −7.305 28.596 1.00 0.01 ATOM 3712 N PHE A 411 0.744 −6.451 29.403 1.00 0.01 ATOM 3714 CA PHE A 411 0.617 −5.191 28.656 1.00 0.01 ATOM 3715 CB PHE A 411 0.284 −4.049 29.619 1.00 0.01 ATOM 3716 CG PHE A 411 1.295 −3.780 30.733 1.00 0.01 ATOM 3717 CD1 PHE A 411 2.346 −2.897 30.519 1.00 0.01 ATOM 3718 CE1 PHE A 411 3.263 −2.650 31.531 1.00 0.00 ATOM 3719 CZ PHE A 411 3.126 −3.277 32.761 1.00 0.01 ATOM 3720 CE2 PHE A 411 2.068 −4.147 32.981 1.00 0.01 ATOM 3721 CD2 PHE A 411 1.150 −4.394 31.970 1.00 0.00 ATOM 3722 C PHE A 411 1.860 −4.851 27.829 1.00 0.00 ATOM 3723 O PHE A 411 2.999 −5.206 28.160 1.00 0.02 ATOM 3724 N LEU A 412 1.605 −4.136 26.747 1.00 0.00 ATOM 3726 CA LEU A 412 2.650 −3.710 25.810 1.00 0.01 ATOM 3727 CB LEU A 412 1.984 −3.656 24.432 1.00 0.51 ATOM 3728 CG LEU A 412 2.939 −3.528 23.245 1.00 0.86 ATOM 3729 CD1 LEU A 412 2.345 −4.205 22.018 1.00 1.15 ATOM 3730 CD2 LEU A 412 3.308 −2.081 22.925 1.00 0.92 ATOM 3731 C LEU A 412 3.213 −2.348 26.222 1.00 0.01 ATOM 3732 O LEU A 412 2.461 −1.415 26.530 1.00 0.01 ATOM 3733 N MET A 413 4.531 −2.250 26.239 1.00 0.01 ATOM 3735 CA MET A 413 5.195 −0.983 26.574 1.00 0.01 ATOM 3736 CB MET A 413 6.310 −1.226 27.579 1.00 1.42 ATOM 3737 CG MET A 413 5.775 −1.694 28.923 1.00 0.91 ATOM 3738 SD MET A 413 7.002 −1.688 30.246 1.00 1.58 ATOM 3739 CE MET A 413 7.448 0.062 30.175 1.00 1.14 ATOM 3740 C MET A 413 5.794 −0.307 25.345 1.00 0.01 ATOM 3741 O MET A 413 6.388 −0.960 24.477 1.00 0.01 ATOM 3742 N MET A 414 5.672 1.008 25.312 1.00 0.00 ATOM 3744 CA MET A 414 6.255 1.792 24.217 1.00 0.01 ATOM 3745 CB MET A 414 5.197 1.954 23.136 1.00 0.42 ATOM 3746 CG MET A 414 5.704 2.789 21.970 1.00 1.17 ATOM 3747 SD MET A 414 4.608 2.810 20.538 1.00 1.20 ATOM 3748 CE MET A 414 3.065 3.254 21.366 1.00 1.92 ATOM 3749 C MET A 414 6.756 3.160 24.681 1.00 0.01 ATOM 3750 O MET A 414 5.983 4.113 24.830 1.00 0.00 ATOM 3751 N ILE A 415 8.056 3.254 24.884 1.00 0.01 ATOM 3753 CA ILE A 415 8.657 4.526 25.303 1.00 0.01 ATOM 3754 CB ILE A 415 9.842 4.231 26.216 1.00 0.24 ATOM 3755 CG2 ILE A 415 10.545 5.519 26.637 1.00 0.37 ATOM 3756 CG1 ILE A 415 9.380 3.452 27.440 1.00 0.21 ATOM 3757 CD1 ILE A 415 10.537 3.177 28.391 1.00 0.15 ATOM 3758 C ILE A 415 9.107 5.320 24.080 1.00 0.01 ATOM 3759 O ILE A 415 10.024 4.907 23.359 1.00 0.01 ATOM 3760 N THR A 416 8.448 6.441 23.843 1.00 0.01 ATOM 3762 CA THR A 416 8.748 7.254 22.658 1.00 0.01 ATOM 3763 CB THR A 416 7.446 7.408 21.881 1.00 0.52 ATOM 3764 OG1 THR A 416 6.945 6.101 21.629 1.00 1.01 ATOM 3765 CG2 THR A 416 7.639 8.117 20.543 1.00 1.12 ATOM 3766 C THR A 416 9.325 8.625 23.019 1.00 0.01 ATOM 3767 O THR A 416 8.744 9.378 23.811 1.00 0.01 ATOM 3768 N ASN A 417 10.471 8.931 22.433 1.00 0.01 ATOM 3770 CA ASN A 417 11.125 10.229 22.628 1.00 0.00 ATOM 3771 CB ASN A 417 12.543 10.132 22.075 1.00 0.31 ATOM 3772 CG ASN A 417 13.254 11.475 22.170 1.00 0.35 ATOM 3773 OD1 ASN A 417 13.106 12.332 21.287 1.00 0.53 ATOM 3774 ND2 ASN A 417 13.993 11.651 23.247 1.00 0.80 ATOM 3777 C ASN A 417 10.368 11.324 21.886 1.00 0.01 ATOM 3778 O ASN A 417 10.458 11.428 20.654 1.00 0.01 ATOM 3779 N LYS A 418 9.838 12.264 22.652 1.00 0.01 ATOM 3781 CA LYS A 418 8.989 13.311 22.075 1.00 0.01 ATOM 3782 CB LYS A 418 7.784 13.572 22.980 1.00 0.76 ATOM 3783 CG LYS A 418 8.047 14.546 24.124 1.00 1.20 ATOM 3784 CD LYS A 418 6.753 14.809 24.889 1.00 1.77 ATOM 3785 CE LYS A 418 6.773 16.139 25.636 1.00 2.20 ATOM 3786 NZ LYS A 418 7.850 16.191 26.631 1.00 2.37 ATOM 3787 C LYS A 418 9.760 14.602 21.778 1.00 0.01 ATOM 3788 O LYS A 418 9.154 15.632 21.459 1.00 0.02 ATOM 3789 N ALA A 419 11.081 14.546 21.869 1.00 0.01 ATOM 3791 CA ALA A 419 11.903 15.688 21.458 1.00 0.00 ATOM 3792 CB ALA A 419 13.230 15.650 22.207 1.00 0.15 ATOM 3793 C ALA A 419 12.154 15.589 19.955 1.00 0.00 ATOM 3794 O ALA A 419 12.447 16.585 19.283 1.00 0.01 ATOM 3795 N THR A 420 12.051 14.367 19.461 1.00 0.01 ATOM 3797 CA THR A 420 11.991 14.108 18.023 1.00 0.01 ATOM 3798 CB THR A 420 13.262 13.400 17.537 1.00 0.00 ATOM 3799 OG1 THR A 420 13.774 12.554 18.563 1.00 0.01 ATOM 3800 CG2 THR A 420 14.365 14.406 17.226 1.00 0.01 ATOM 3801 C THR A 420 10.714 13.325 17.713 1.00 0.01 ATOM 3802 O THR A 420 9.651 13.924 17.516 1.00 0.01 ATOM 3803 N ASP A 421 10.836 12.009 17.632 1.00 0.01 ATOM 3805 CA ASP A 421 9.695 11.108 17.393 1.00 0.01 ATOM 3806 CB ASP A 421 9.064 11.381 16.023 1.00 2.55 ATOM 3807 CG ASP A 421 10.108 11.518 14.905 1.00 2.84 ATOM 3808 OD1 ASP A 421 11.024 10.706 14.858 1.00 3.45 ATOM 3809 OD2 ASP A 421 10.035 12.509 14.201 1.00 2.99 ATOM 3810 C ASP A 421 10.143 9.649 17.489 1.00 0.01 ATOM 3811 O ASP A 421 9.368 8.722 17.223 1.00 0.01 ATOM 3812 N GLY A 422 11.387 9.459 17.893 1.00 0.01 ATOM 3814 CA GLY A 422 11.985 8.120 17.874 1.00 0.01 ATOM 3815 C GLY A 422 11.553 7.251 19.047 1.00 0.00 ATOM 3816 O GLY A 422 11.748 7.617 20.212 1.00 0.00 ATOM 3817 N ILE A 423 10.880 6.158 18.733 1.00 0.01 ATOM 3819 CA ILE A 423 10.570 5.162 19.763 1.00 0.00 ATOM 3820 CB ILE A 423 9.661 4.089 19.179 1.00 0.37 ATOM 3821 CG2 ILE A 423 9.305 3.046 20.234 1.00 0.32 ATOM 3822 CG1 ILE A 423 8.393 4.706 18.609 1.00 0.51 ATOM 3823 CD1 ILE A 423 7.475 3.623 18.059 1.00 0.70 ATOM 3824 C ILE A 423 11.871 4.534 20.242 1.00 0.01 ATOM 3825 O ILE A 423 12.566 3.841 19.486 1.00 0.01 ATOM 3826 N LEU A 424 12.153 4.741 21.515 1.00 0.01 ATOM 3828 CA LEU A 424 13.420 4.322 22.094 1.00 0.01 ATOM 3829 CB LEU A 424 13.697 5.172 23.330 1.00 0.01 ATOM 3830 CG LEU A 424 13.710 6.667 23.041 1.00 0.01 ATOM 3831 CD1 LEU A 424 13.732 7.462 24.342 1.00 0.01 ATOM 3832 CD2 LEU A 424 14.884 7.057 22.149 1.00 0.01 ATOM 3833 C LEU A 424 13.331 2.880 22.544 1.00 0.01 ATOM 3834 O LEU A 424 14.291 2.125 22.369 1.00 0.01 ATOM 3835 N PHE A 425 12.153 2.486 23.003 1.00 0.01 ATOM 3837 CA PHE A 425 11.976 1.137 23.557 1.00 0.01 ATOM 3838 CB PHE A 425 12.132 1.207 25.071 1.00 0.01 ATOM 3839 CG PHE A 425 13.530 1.519 25.595 1.00 0.01 ATOM 3840 CD1 PHE A 425 14.590 0.672 25.298 1.00 0.01 ATOM 3841 CE1 PHE A 425 15.858 0.949 25.792 1.00 0.01 ATOM 3842 CZ PHE A 425 16.065 2.071 26.583 1.00 0.01 ATOM 3843 CE2 PHE A 425 15.004 2.917 26.879 1.00 0.01 ATOM 3844 CD2 PHE A 425 13.737 2.640 26.387 1.00 0.01 ATOM 3845 C PHE A 425 10.607 0.524 23.279 1.00 0.00 ATOM 3846 O PHE A 425 9.567 1.094 23.639 1.00 0.01 ATOM 3847 N LEU A 426 10.635 −0.688 22.751 1.00 0.00 ATOM 3849 CA LEU A 426 9.418 −1.500 22.604 1.00 0.01 ATOM 3850 CB LEU A 426 9.215 −1.912 21.153 1.00 0.28 ATOM 3851 CG LEU A 426 8.600 −0.791 20.330 1.00 0.43 ATOM 3852 CD1 LEU A 426 8.368 −1.251 18.898 1.00 0.63 ATOM 3853 CD2 LEU A 426 7.281 −0.350 20.949 1.00 1.00 ATOM 3854 C LEU A 426 9.490 −2.750 23.477 1.00 0.01 ATOM 3855 O LEU A 426 10.476 −3.502 23.442 1.00 0.01 ATOM 3856 N GLY A 427 8.422 −2.987 24.219 1.00 0.01 ATOM 3858 CA GLY A 427 8.407 −4.123 25.141 1.00 0.01 ATOM 3859 C GLY A 427 7.051 −4.798 25.346 1.00 0.00 ATOM 3860 O GLY A 427 5.997 −4.354 24.873 1.00 0.01 ATOM 3861 N LYS A 428 7.120 −5.898 26.072 1.00 0.00 ATOM 3863 CA LYS A 428 5.944 −6.689 26.445 1.00 0.01 ATOM 3864 CB LYS A 428 5.789 −7.862 25.484 1.00 0.01 ATOM 3865 CG LYS A 428 4.595 −8.725 25.884 1.00 0.01 ATOM 3866 CD LYS A 428 4.667 −10.117 25.270 1.00 0.00 ATOM 3867 CE LYS A 428 4.692 −10.060 23.750 1.00 0.00 ATOM 3868 NZ LYS A 428 4.758 −11.416 23.186 1.00 0.01 ATOM 3869 C LYS A 428 6.127 −7.261 27.844 1.00 0.01 ATOM 3870 O LYS A 428 6.958 −8.157 28.051 1.00 0.01 ATOM 3871 N VAL A 429 5.333 −6.782 28.784 1.00 0.00 ATOM 3873 CA VAL A 429 5.387 −7.325 30.142 1.00 0.01 ATOM 3874 CB VAL A 429 5.105 −6.215 31.144 1.00 0.01 ATOM 3875 CG1 VAL A 429 5.108 −6.750 32.572 1.00 0.00 ATOM 3876 CG2 VAL A 429 6.132 −5.101 30.998 1.00 0.01 ATOM 3877 C VAL A 429 4.368 −8.447 30.294 1.00 0.00 ATOM 3878 O VAL A 429 3.173 −8.211 30.519 1.00 0.01 ATOM 3879 N GLU A 430 4.857 −9.666 30.153 1.00 0.02 ATOM 3881 CA GLU A 430 4.000 −10.843 30.270 1.00 0.01 ATOM 3882 CB GLU A 430 4.477 −11.877 29.261 1.00 0.30 ATOM 3883 CG GLU A 430 3.550 −13.085 29.206 1.00 0.57 ATOM 3884 CD GLU A 430 4.321 −14.244 28.598 1.00 0.25 ATOM 3885 OE1 GLU A 430 3.692 −15.219 28.209 1.00 0.58 ATOM 3886 OE2 GLU A 430 5.534 −14.216 28.754 1.00 0.26 ATOM 3887 C GLU A 430 4.096 −11.422 31.675 1.00 0.02 ATOM 3888 O GLU A 430 3.177 −12.097 32.153 1.00 0.01 ATOM 3889 N ASN A 431 5.220 −11.149 32.313 1.00 0.00 ATOM 3891 CA ASN A 431 5.443 −11.521 33.713 1.00 0.00 ATOM 3892 CB ASN A 431 5.740 −13.018 33.801 1.00 0.00 ATOM 3893 CG ASN A 431 5.920 −13.475 35.253 1.00 0.01 ATOM 3894 OD1 ASN A 431 6.693 −12.891 36.026 1.00 0.02 ATOM 3895 ND2 ASN A 431 5.257 −14.566 35.584 1.00 0.02 ATOM 3898 C ASN A 431 6.632 −10.736 34.246 1.00 0.02 ATOM 3899 O ASN A 431 7.777 −11.192 34.119 1.00 0.01 ATOM 3900 N PRO A 432 6.349 −9.732 35.060 1.00 0.00 ATOM 3901 CA PRO A 432 7.387 −8.783 35.483 1.00 0.02 ATOM 3902 CB PRO A 432 6.637 −7.649 36.110 1.00 0.02 ATOM 3903 CG PRO A 432 5.164 −8.012 36.211 1.00 0.01 ATOM 3904 CD PRO A 432 5.016 −9.365 35.537 1.00 0.02 ATOM 3905 C PRO A 432 8.408 −9.346 36.479 1.00 0.01 ATOM 3906 O PRO A 432 9.515 −8.805 36.553 1.00 0.00 ATOM 3907 N THR A 433 8.145 −10.500 37.076 1.00 0.02 ATOM 3909 CA THR A 433 9.086 −11.048 38.054 1.00 0.01 ATOM 3910 CB THR A 433 8.340 −11.865 39.104 1.00 0.02 ATOM 3911 OG1 THR A 433 7.900 −13.083 38.520 1.00 0.01 ATOM 3912 CG2 THR A 433 7.136 −11.114 39.660 1.00 0.01 ATOM 3913 C THR A 433 10.127 −11.927 37.369 1.00 0.02 ATOM 3914 O THR A 433 11.161 −12.247 37.969 1.00 0.02 ATOM 3915 N LYS A 434 9.910 −12.226 36.098 1.00 0.01 ATOM 3917 CA LYS A 434 10.908 −12.974 35.324 1.00 0.02 ATOM 3918 CB LYS A 434 10.202 −13.816 34.268 1.00 2.22 ATOM 3919 CG LYS A 434 9.202 −14.782 34.891 1.00 2.65 ATOM 3920 CD LYS A 434 9.867 −15.756 35.857 1.00 2.62 ATOM 3921 CE LYS A 434 8.831 −16.644 36.534 1.00 3.30 ATOM 3922 NZ LYS A 434 9.470 −17.570 37.481 1.00 3.61 ATOM 3923 C LYS A 434 11.891 −12.016 34.651 1.00 0.02 ATOM 3924 O LYS A 434 11.859 −11.817 33.430 1.00 0.00 ATOM 3925 N SER A 435 12.761 −11.438 35.460 1.00 0.02 ATOM 3927 CA SER A 435 13.747 −10.479 34.960 1.00 0.02 ATOM 3928 CB SER A 435 13.389 −9.101 35.503 1.00 1.31 ATOM 3929 OG SER A 435 12.076 −8.780 35.062 1.00 1.94 ATOM 3930 C SER A 435 15.152 −10.861 35.411 1.00 0.02 ATOM 3931 O SER A 435 15.582 −10.351 36.435 1.00 0.01 ATOM 3932 OXT SER A 435 15.790 −11.618 34.691 1.00 0.01 

1-52. (canceled)
 53. An isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) an isolated polypeptide comprising amino acids 1 to 435 of SEQ ID NO:2; (b) an isolated polypeptide comprising amino acids 2 to 435 of SEQ ID NO:2; and (c) an isolated mature polypeptide comprising amino acids 39 to 435 of SEQ ID NO:2.
 54. The isolated polypeptide of claim 53, wherein said polypeptide is (a).
 55. The isolated polypeptide of claim 53, wherein said polypeptide is (b).
 56. The isolated polypeptide of claim 53, wherein said polypeptide is (c).
 57. An isolated polypeptide produced by a method comprising: (a) culturing an isolated recombinant host cell comprising a vector that comprises the coding region encoding the polypeptide of claim 53 under conditions such that the polypeptide of claim 53 is expressed; and (b) recovering said polypeptide.
 58. The isolated polynucleotide of claim 53 wherein said polypeptide sequence further comprises a heterologous polypeptide sequence.
 59. The isolated polynucleotide of claim 58 wherein said heterologous polypeptide is the Fc domain of immunoglobulin.
 60. An isolated polypeptide comprising the polypeptide encoded by the cDNA clone contained in ATCC Deposit No: PTA-2766.
 61. The isolated polypeptide of claim 53(a), (b), or (c), wherein said polypeptide has serine protease inhibitor activity with Arg/Lys specificity.
 62. The isolated polypeptide of claim 53(a), (b), or (c), wherein said encoded polypeptide comprises one amino acid substitution in the region selected from the group consisting of: a.) amino acids 63 to 80 of SEQ ID NO:2; b.) amino acids 125 to 140 of SEQ ID NO:2; c.) amino acids 185 to 209 of SEQ ID NO:2; d.) amino acids 217 to 227 of SEQ ID NO:2; e.) amino acids 306 to 315 of SEQ ID NO:2; f.) amino acids 368 to 377 of SEQ ID NO:2; g.) amino acid 405 to 415 of SEQ ID NO:2; and wherein said polypeptide has serine protease inhibitor activity with Arg/Lys specificity, or is catalytically inactive yet retains ability to bind to a serine protease substrate. 